July 2018
Volume 59, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2018
Protein structural prediction of modified Volvox channelrhodopsin-1 and the verification by amino acid sequence
Author Affiliations & Notes
  • Eriko Sugano
    Chemistry and Biological Sciences, Iwate Univ., Morioka, Iwate, Japan
  • Kitako Tabata
    Chemistry and Biological Sciences, Iwate Univ., Morioka, Iwate, Japan
  • Yuko Sakajiri
    Chemistry and Biological Sciences, Iwate Univ., Morioka, Iwate, Japan
  • Yoshito Watanabe
    Chemistry and Biological Sciences, Iwate Univ., Morioka, Iwate, Japan
  • Makoto Tamai
    Tohoku Univ. Hospital, Sendai, Japan
  • Hiroshi Tomita
    Chemistry and Biological Sciences, Iwate Univ., Morioka, Iwate, Japan
  • Footnotes
    Commercial Relationships   Eriko Sugano, None; Kitako Tabata, None; Yuko Sakajiri, None; Yoshito Watanabe, None; Makoto Tamai, None; Hiroshi Tomita, None
  • Footnotes
    Support  KAKENHI No. 16H05485, 16K15729 and 16K11314, Iwate Research program
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 3081. doi:
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      Eriko Sugano, Kitako Tabata, Yuko Sakajiri, Yoshito Watanabe, Makoto Tamai, Hiroshi Tomita; Protein structural prediction of modified Volvox channelrhodopsin-1 and the verification by amino acid sequence. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3081.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : We previously designed a modified vorvox channelrhdopsin-1(mVChR1) protein chimera which has the broader action than that of Chlamydomonas channelrhodopin-2(ChR2). To improve the ion influx of mVChR1, protein structural prediction was performed. To clarify mature mVChR1 protein structure, we investigated the N-terminal amino acid sequence of mVChR1.

Methods : To study the ion transport route, we predict the three dimensional structure of mVChR1 by Swiss modeling. The secondary protein structure and full-chain protein structure was predicted by TMHMM and Robetta beta model, respectively. To reveal the N-terminal amino acid sequence of mVChR1 protein, AAV-CAG-mVChR1-Venus plasmid vector was transfected into HEK293 cells using DNA-Ca phosphate co-precipitation method. After 3days of cultivation, mVChR1 protein expression in the membrane was confirmed under a fluorescent microscopy and the protein was extracted. mVChR1 protein was collected by immunoprecipitation using venus-tag and N-terminal amino acid sequence was analyzed (Hokkaido system science, Japan).

Results : Three dimensional structure of mVChR1 was compared to that of ChR2 and Vorvox channelrodopsin-1 (VChR1) by ClustaIW analysis. These results showed that N-terminal structure had low homology among these proteins. Predictive analysis by TMHMM and Robetta beta model showed that mVChR1 exists as an 8-transmembrane protein. However, analysis of N-terminal amino acid sequence reveled that mature mVChR1 was a 7-transmemberane protein in the cells.

Conclusions : In the design of mVChR1, N-terminal of VChR1 was modified to improve the membrane translocation with consideration of previous reports. We have supposed that N-terminal of mVChR1 should receive the processing at the translocation to the membrane. However, protein structural analysis predicted that N-terminal signal exists after the translocation. Contrary to this prediction, amino acid sequence showed that N-terminal signal receive the processing and mature mVChR1 is a 7-transmemberane protein. These results indicated that N-terminal signal is a key amino acids to translocate the protein to the cellular membrane.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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