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Characterization of pH Dependent Properties of mCherry Mutant, I202T

형광 단백질 mCherry-I202T의 pH 감응성 분석

  • Lee, Sangmin (Department of Chemical Engineering, Hongik University) ;
  • Chung, Minsub (Department of Chemical Engineering, Hongik University)
  • 이상민 (홍익대학교 화학공학과) ;
  • 정민섭 (홍익대학교 화학공학과)
  • Received : 2020.11.19
  • Accepted : 2020.12.11
  • Published : 2021.02.10

Abstract

mCherry is one of the well-understood red fluorescent proteins which has a similar tertiary structure as GFPs, but pH resistant due to the lack of hydrogen bond network. Whereas mCherry-I202T showed far-red fluorescence and also pH sensitive property because of the additional hydrogen bond formed by substituting Ile of 202 amino acid sequence on mCherry with Thr. In order to verify the pH sensitive characteristic of mCherry-I202T owing to the extension of hydrogen bond, UV-vis spectrum was measured over the range of acidic to basic pH. We also demonstrate further possibilities of applying mCherry-I202T as a pH sensor.

DsRed에서 유래한 적색 형광 단백질들 중 하나인 mCherry는 GFP와 유사한 3차 구조를 가진 잘 알려진 적색 형광 단백질 중 하나이며, 수소결합 네트워크를 형성하고 있지 않아서 pH 변화에 민감하지 않다. 반면 mCherry의 발색단 근처에서 돌연변이를 야기 시켜 만든 mCherry-I202T는 mCherry 단백질의 202번째 아미노산인 이소루신(Ile)을 트레오닌(Thr)으로 치환함으로써 모체와는 다르게 추가적인 수소결합을 형성하여, 주위 pH에 더욱 민감하게 반응할 뿐 아니라 적색 편이된 형광을 보였다. 수소결합이 확장된 I202T의 pH 민감성을 검증하기 위해 산성과 염기성 pH 범위에서 I202T의 UV-vis 흡광 스펙트럼 변화와 가역성을 확인하고, 그를 pH sensor에 적용 가능한지 그 가능성을 검증하고자 하였다.

Keywords

References

  1. N. C. Shaner, R. E. Campbell, P. A. Steinbach, B. N. Giepmans, A. E. Palmer, and R. Y. Tsien, Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein, Nat. Biotechnol., 22, 1567-1572 (2004). https://doi.org/10.1038/nbt1037
  2. R. E. Campbell, A. E. Palmer, P. A. Steinbach, G. S. Baird, D. A. Zacharias, and R. Y. Tsien, A monomeric red fluorescent protein, Proc. Natl. Acad. Sci. U.S.A., 7877-7882 (2002).
  3. G. J. Bevis and B. S. Brooke, Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed), Nat. Biotechnol., 20(1), 83-87 (2002). https://doi.org/10.1038/nbt0102-83
  4. X. Shu, N. C. Shaner, C. A. Yarbrough, R. Y. Tsien, and S. J. Remington. Novel chromophores and buried charges control color in mFruits, Biochemistry, 45, 9639-9647 (2006). https://doi.org/10.1021/bi060773l
  5. R. Bizzarri, C. Arcangeli, D. Arosio, F. Ricci, P. Faraci, F. Cardarelli, and F. Beltram, Development of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies. Biophys. J., 90(9), 3300-3314 (2006). https://doi.org/10.1529/biophysj.105.074708
  6. M. Pereira, D. Tome, A. S. Domingues, A. S. Varanda, C. Paulo, M. A. Santos, and A. R. Soares, A fluorescence-based sensor assay that monitors general protein aggregation in human cells, Biotechnol. J., 13(4), 1700676 (2018). https://doi.org/10.1002/biot.201700676
  7. Y. Kim, K. Song, H. Lee, D. Kim, J. Kim, and M. Chung, Single amino acid replacement transforms mCherry to a far-red fluorescent protein, Biotechnol. Bioprocess Eng., 21(6), 720-725 (2016). https://doi.org/10.1007/s12257-016-0402-3
  8. G. S. Baird, D. A. Zacharias, and R. Y. Tsien, Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. U.S.A., 97(22), 11984-11989 (2000). https://doi.org/10.1073/pnas.97.22.11984