Abstract
Genetically encoded fluorescent proteins are extensively utilized for labeling and imaging proteins, organelles, cell tissues, and whole organisms. In this study, we explored the feasibility of mRFP1 and its variants for measuring intracellular temperature. A linear relationship was observed between the temperature and fluorescence intensity of mRFP1 and its variants. Temperature sensitivities of E. coli expressing mRFP1, mRFP-P63A and mRFP-P63A[(4R)-FP] were −1.27%, −1.26% and −0.77%/°C, respectively. Finally, we demonstrated the potentiality of mRFP1 and its variants as an in vivo temperature sensor.
Keywords: temperature sensor, mRFP1, 4-fluoroproline, non-canonical amino acid incorporation, thermal stability
References
- 1.DeBerardinis R. J., Lum J. J., Hatzivassiliou G., Thompson C. B. The biology of cancer: Metabolic reprogramming fuels cell growth and proliferation. Cell Metab. 2008;7:11–20. doi: 10.1016/j.cmet.2007.10.002. [DOI] [PubMed] [Google Scholar]
- 2.Nakamura T., Matsuoka I. Calorimetric studies of heat of respiration of mitochondria. J. Biochem. 1978;84:39–46. doi: 10.1093/oxfordjournals.jbchem.a132117. [DOI] [PubMed] [Google Scholar]
- 3.Lowell B. B., Spiegelman B. M. Towards a molecular understanding of adaptive thermogenesis. Nature. 2000;404:652–660. doi: 10.1038/35007527. [DOI] [PubMed] [Google Scholar]
- 4.Kallerhoff M., Karnebogen M., Singer D., Dettenbach A., Gralher U., Ringert R. H. Microcalorimetric measurements carried out on isolated tumorous and nontumorous tissue samples from organs in the urogenital tract in comparison to histological and impulse-cytophotometric investigations. Urol. Res. 1996;24:83–91. doi: 10.1007/BF00431084. [DOI] [PubMed] [Google Scholar]
- 5.Ye F., Wu C., Jin Y., Chan Y. H., Zhang X., Chiu D. T. Ratiometric temperature sensing with semiconducting polymer dots. J. Am. Chem. Soc. 2011;133:8146–8149. doi: 10.1021/ja202945g. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Chen Y. Y., Wood A. W. Application of a temperature-dependent fluorescent dye (Rhodamine B) to the measurement of radiofrequency radiation-induced temperature changes in biological samples. Bioelectromagnetics. 2009;30:583–590. doi: 10.1002/bem.20514. [DOI] [PubMed] [Google Scholar]
- 7.Yang J. M., Yang H., Lin L. Quantum dot nano thermometers reveal heterogeneous local thermogenesis in living cells. ACS Nano. 2011;5:5067–5071. doi: 10.1021/nn201142f. [DOI] [PubMed] [Google Scholar]
- 8.Biju V., Makita Y., Sonoda A., Yokoyama H., Baba Y., Ishikawa M. Temperature-sensitive photoluminescence of CdSe quantum dot clusters. J. Phys. Chem. B. 2005;109:13899–13905. doi: 10.1021/jp050424l. [DOI] [PubMed] [Google Scholar]
- 9.Vetrone F., Naccache R., Zamarrón A., Juarranz de la Fuente A., Sanz-Rodríguez F., Martinez Maestro L., Martín Rodriguez E., Jaque D., García Solé J., Capobianco J. A. Temperature sensing using fluorescent nanothermometers. ACS Nano. 2010;4:3254–3258. doi: 10.1021/nn100244a. [DOI] [PubMed] [Google Scholar]
- 10.Gota C., Okabe K., Funatsu T., Harada Y., Uchiyama S. Hydrophilic fluorescent nanogel thermometer for intracellular thermometry. J. Am. Chem. Soc. 2009;131:2766–2767. doi: 10.1021/ja807714j. [DOI] [PubMed] [Google Scholar]
- 11.Albers A. E., Chan E. M., McBride P. M., Ajo-Franklin C. M., Cohen B. E., Helms B. A. Dual-emitting quantum dot/quantum rod-based nanothermometers with enhanced response and sensitivity in live cells. J. Am. Chem. Soc. 2012;134:9565–9568. doi: 10.1021/ja302290e. [DOI] [PubMed] [Google Scholar]
- 12.McLaurin E. J., Vlaskin V. A., Gamelin D. R. Water-soluble dual-emitting nanocrystals for ratiometric optical thermometry. J. Am. Chem. Soc. 2011;133:14978–14980. doi: 10.1021/ja206956t. [DOI] [PubMed] [Google Scholar]
- 13.Donner J. S., Thompson S. A., Kreuzer M. P., Baffou G., Quidant R. Mapping intracellular temperature using green fluorescent protein. Nano Lett. 2012;12:2107–2111. doi: 10.1021/nl300389y. [DOI] [PubMed] [Google Scholar]
- 14.Donner J. S., Thompson S. A., Ortega C. A., Morales J., Rico L. G., Santos S. I. C. O., Quidant R. Imaging of plasmonic heating in a living organism. ACS Nano. 2013;7:8666–8672. doi: 10.1021/nn403659n. [DOI] [PubMed] [Google Scholar]
- 15.Wong F. H., Banks D. S., Abu-Arish A., Fradin C. A molecular thermometer based on fluorescent protein blinking. J. Am. Chem. Soc. 2007;129:10302–10303. doi: 10.1021/ja0715905. [DOI] [PubMed] [Google Scholar]
- 16.Leiderman P., Huppert D., Agmon N. Transition in the temperature-dependence of GFP fluorescence: from proton wires to proton exit. Biophys. J. 2006;90:1009–1018. doi: 10.1529/biophysj.105.069393. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Okabe K., Inada N., Gota C., Harada Y., Funatsu T., Uchiyama S. Intracellular temperature mapping with a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy. Nat. Commun. 2012;3:1–8. doi: 10.1038/ncomms1714. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Chudakov D. M., Matz M. V., Lukyanov S., Lukyanov K. A. Fluorescent proteins and their applications in imaging living cells and tissues. Physiol. Rev. 2010;90:1103–1163. doi: 10.1152/physrev.00038.2009. [DOI] [PubMed] [Google Scholar]
- 19.Shcherbakova D. M., Subach O. M., Verkhusha V. V. Red fluorescent proteins: Advanced imaging applications and future design. Angew. Chem. Int. Ed. 2012;51:10724–10738. doi: 10.1002/anie.201200408. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Raghunathan G., Sokalingam S., Soundrarajan N., Munussami G., Madan B., Lee S. A comparative study on the stability and structure of two different green fluorescent proteins in organic co-solvent systems. Biotechnol. Bioproc. Eng. 2013;18:342–349. doi: 10.1007/s12257-012-0579-z. [DOI] [Google Scholar]
- 21.Deepankumar K., Nadarajan S. P., Ayyadurai N., Yun H. Enhancing the biophysical properties of mRFP1 through incorporation of fluoroproline. Biochem. Biophys. Res. Commun. 2013;440:509–514. doi: 10.1016/j.bbrc.2013.09.062. [DOI] [PubMed] [Google Scholar]
- 22.Zelus B. D., Schickli J. H., Blau D. M., Weiss S. R., Holmes K. V. Conformational changes in the spike glycoprotein of murine coronavirus are induced at 37 degrees C either by soluble murine CEACAM1 receptors or by pH 8. J. Virol. 2003;77:830–840. doi: 10.1128/JVI.77.2.830-840.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Azim-Zadeh O., Hillebrecht A., Linne U., Marahiel M. A., Klebe G., Lingelbach K., Nyalwidhe J. Use of biotin derivatives to probe conformational changes in proteins. J. Biol. Chem. 2007;282:21609–21617. doi: 10.1074/jbc.M610921200. [DOI] [PubMed] [Google Scholar]
- 24.Haake C. H. The significance of the temperature dependence of fluorescence intensity. J. Electrochem. Soc. 1961;108:78–82. doi: 10.1149/1.2428016. [DOI] [Google Scholar]
- 25.Ross D., Gaitan M., Locascio L. E. Temperature measurement in microfluidic systems using a temperature-dependent fluorescent dye. Anal. Chem. 2001;73:4117–4123. doi: 10.1021/ac010370l. [DOI] [PubMed] [Google Scholar]
- 26.Chen C. Y., Chen C. T. A PNIPAM-based fluorescent nanothermometer with ratiometric readout. Chem. Commun. 2011;47:994–996. doi: 10.1039/C0CC04450D. [DOI] [PubMed] [Google Scholar]
- 27.Yin L., He C., Huang C., Zhu W., Wang X., Xu Y., Qian X. A dual pH and temperature responsive polymeric fluorescent sensor and its imaging application in living cells. Chem. Commun. 2012;48:4486–4488. doi: 10.1039/c2cc30404j. [DOI] [PubMed] [Google Scholar]
- 28.Budisa N. Prolegomena to future experimental efforts on genetic code engineering by expanding its amino acid repertoire. Angew. Chem. Int. Ed. 2004;43:6426–6463. doi: 10.1002/anie.200300646. [DOI] [PubMed] [Google Scholar]
- 29.Johnson J. A., Lu Y. Y., Van Deventer J. A., Tirrell D. A. Residue-specific incorporation of non-canonical amino acids into proteins: Recent developments and applications. Curr. Opin. Chem. Biol. 2010;14:774–780. doi: 10.1016/j.cbpa.2010.09.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Zheng S., Kwon I. Manipulation of enzyme properties by noncanonical amino acid incorporation. Biotechno. J. 2012;7:47–60. doi: 10.1002/biot.201100267. [DOI] [PubMed] [Google Scholar]
- 31.Holzberger B., Marx A. Replacing 32 proline residues by a noncanonical amino acid results in a highly active DNA polymerase. J. Am. Chem. Soc. 2010;132:15708–15713. doi: 10.1021/ja106525y. [DOI] [PubMed] [Google Scholar]
- 32.Edwardraja S., Sriram S., Govindan R., Budisa N., Lee S. G. Enhancing the thermal stability of a single-chain Fv fragment by in vivo global fluorination of the proline residues. Mol. Biosyst. 2011;7:258–265. doi: 10.1039/C0MB00154F. [DOI] [PubMed] [Google Scholar]
- 33.Crespo M. D., Rubini M. Rational design of protein stability: Effect of (2S,4R)-4-fluoroproline on the stability and folding pathway of ubiquitin. PLoS One. 2011;6:e19425. doi: 10.1371/journal.pone.0019425. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Baker P. J., Montclare J. K. Enhanced refoldability and thermoactivity of fluorinated phosphotriesterase. ChemBio-Chem. 2011;12:1845–1848. doi: 10.1002/cbic.201100221. [DOI] [PubMed] [Google Scholar]
- 35.Hoesl M. G., Acevedo-Rocha C. G., Nehring S., Royter M., Wolschner C., Wiltschi B., Budisa N., Antranikian G. Lipase congeners designed by genetic code engineering. Chem-CatChem. 2011;3:213–221. [Google Scholar]
- 36.Acevedo-Rocha C. G., Hoesl M. G., Nehring S., Royter M., Wolschner C., Wiltschi B., Antranikian G., Budisa N. Non-canonical amino acids as a useful synthetic biological tool for lipase-catalysed reactions in hostile environments. Catal. Sci. Technol. 2013;3:1198–1201. doi: 10.1039/c3cy20712a. [DOI] [Google Scholar]
- 37.Ayyadurai N., Deepankumar K., Prabhu N. S., Budisa N., Yun H. Evaluation and biosynthetic incorporation of chlorotyrosine into recombinant Proteins. Biotechnol. Bioproc. Eng. 2012;17:679–686. doi: 10.1007/s12257-012-0066-6. [DOI] [Google Scholar]