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Functional and fluorescence analyses of tryptophan residues in H-pyrophosphatase of Clostridium tetani.

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Title: Functional and fluorescence analyses of tryptophan residues in H-pyrophosphatase of Clostridium tetani.
Authors: Chen, Yen-Wei1, Lee, Ching-Hung1, Huang, Yun-Tzu1, Pan, Yih-Jiuan1, Lin, Shih-Ming1, Lo, Yueh-Yu1, Lee, Chien-Hsien1, Huang, Lin-Kun1, Huang, Yu-Fen1, Hsu, Yu-Di1, Pan, Rong-Long1 rlpan@life.nthu.edu.tw
Source: Journal of Bioenergetics & Biomembranes. Apr2014, Vol. 46 Issue 2, p127-134. 8p.
Subjects: Fluorimetry, Tryptophan, Inorganic pyrophosphatase, Clostridium tetani, Site-specific mutagenesis, Hydrolysis, Proton transfer reactions
Abstract: Homodimeric proton-translocating pyrophosphatase (H-PPase; EC 3.6.1.1) maintains the cytoplasmic pH homeostasis of many bacteria and higher plants by coupling pyrophosphate (PP) hydrolysis and proton translocation. H-PPase accommodates several essential motifs involved in the catalytic mechanism, including the PP binding motif and Acidic I and II motifs. In this study, 3 intrinsic tryptophan residues, Trp-75, Trp-365, and Trp-602, in H-PPase from Clostridium tetani were used as internal probes to monitor the local conformational state of the periplasm domain, transmembrane region, and cytoplasmic domain, respectively. Upon binding of the substrate analog Mg-imidodiphosphate (Mg-IDP), local structural changes prevented the modification of tryptophan residues by N-bromosuccinimide (NBS), especially at Trp-602. Following Mg-P binding, Trp-75 and Trp-365, but not Trp-602, were slightly protected from structural modifications by NBS. These results reveal the conformation of H-PPase is distinct in the presence of different ligands. Moreover, analyses of the Stern-Volmer relationship and steady-state fluorescence anisotropy also indicate that the local structure around Trp-602 is more exposed to solvent and varied under different environments. In addition, Trp-602 was identified to be a crucial residue in the H-PPase that may potentially be involved in stabilizing the structure of the catalytic region by site-directed mutagenesis analysis. [ABSTRACT FROM AUTHOR]
Copyright of Journal of Bioenergetics & Biomembranes is the property of Springer Nature and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
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  Data: Functional and fluorescence analyses of tryptophan residues in H-pyrophosphatase of Clostridium tetani.
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  Data: <searchLink fieldCode="AR" term="%22Chen%2C+Yen-Wei%22">Chen, Yen-Wei</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Lee%2C+Ching-Hung%22">Lee, Ching-Hung</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Huang%2C+Yun-Tzu%22">Huang, Yun-Tzu</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Pan%2C+Yih-Jiuan%22">Pan, Yih-Jiuan</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Lin%2C+Shih-Ming%22">Lin, Shih-Ming</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Lo%2C+Yueh-Yu%22">Lo, Yueh-Yu</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Lee%2C+Chien-Hsien%22">Lee, Chien-Hsien</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Huang%2C+Lin-Kun%22">Huang, Lin-Kun</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Huang%2C+Yu-Fen%22">Huang, Yu-Fen</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Hsu%2C+Yu-Di%22">Hsu, Yu-Di</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Pan%2C+Rong-Long%22">Pan, Rong-Long</searchLink><relatesTo>1</relatesTo><i> rlpan@life.nthu.edu.tw</i>
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  Data: <searchLink fieldCode="JN" term="%22Journal+of+Bioenergetics+%26+Biomembranes%22">Journal of Bioenergetics & Biomembranes</searchLink>. Apr2014, Vol. 46 Issue 2, p127-134. 8p.
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  Data: <searchLink fieldCode="DE" term="%22Fluorimetry%22">Fluorimetry</searchLink><br /><searchLink fieldCode="DE" term="%22Tryptophan%22">Tryptophan</searchLink><br /><searchLink fieldCode="DE" term="%22Inorganic+pyrophosphatase%22">Inorganic pyrophosphatase</searchLink><br /><searchLink fieldCode="DE" term="%22Clostridium+tetani%22">Clostridium tetani</searchLink><br /><searchLink fieldCode="DE" term="%22Site-specific+mutagenesis%22">Site-specific mutagenesis</searchLink><br /><searchLink fieldCode="DE" term="%22Hydrolysis%22">Hydrolysis</searchLink><br /><searchLink fieldCode="DE" term="%22Proton+transfer+reactions%22">Proton transfer reactions</searchLink>
– Name: Abstract
  Label: Abstract
  Group: Ab
  Data: Homodimeric proton-translocating pyrophosphatase (H-PPase; EC 3.6.1.1) maintains the cytoplasmic pH homeostasis of many bacteria and higher plants by coupling pyrophosphate (PP) hydrolysis and proton translocation. H-PPase accommodates several essential motifs involved in the catalytic mechanism, including the PP binding motif and Acidic I and II motifs. In this study, 3 intrinsic tryptophan residues, Trp-75, Trp-365, and Trp-602, in H-PPase from Clostridium tetani were used as internal probes to monitor the local conformational state of the periplasm domain, transmembrane region, and cytoplasmic domain, respectively. Upon binding of the substrate analog Mg-imidodiphosphate (Mg-IDP), local structural changes prevented the modification of tryptophan residues by N-bromosuccinimide (NBS), especially at Trp-602. Following Mg-P binding, Trp-75 and Trp-365, but not Trp-602, were slightly protected from structural modifications by NBS. These results reveal the conformation of H-PPase is distinct in the presence of different ligands. Moreover, analyses of the Stern-Volmer relationship and steady-state fluorescence anisotropy also indicate that the local structure around Trp-602 is more exposed to solvent and varied under different environments. In addition, Trp-602 was identified to be a crucial residue in the H-PPase that may potentially be involved in stabilizing the structure of the catalytic region by site-directed mutagenesis analysis. [ABSTRACT FROM AUTHOR]
– Name: AbstractSuppliedCopyright
  Label:
  Group: Ab
  Data: <i>Copyright of Journal of Bioenergetics & Biomembranes is the property of Springer Nature and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.)
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        Value: 10.1007/s10863-013-9532-x
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      – Code: eng
        Text: English
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      – SubjectFull: Fluorimetry
        Type: general
      – SubjectFull: Tryptophan
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      – SubjectFull: Inorganic pyrophosphatase
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      – SubjectFull: Clostridium tetani
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