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Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1
The proteinase K subfamily enzymes, thermophilic Aqualysin I (AQN) from Thermus aquaticus YT-1 and psychrophilic serine protease (VPR) from Vibrio sp. PA-44, have six and seven salt bridges, respectively. To understand the possible significance of salt bridges in the thermal stability of AQN, we pre...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Springer
2014
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4131155/ https://www.ncbi.nlm.nih.gov/pubmed/25136511 http://dx.doi.org/10.1186/s13568-014-0059-2 |
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author | Sakaguchi, Masayoshi Osaku, Kanae Maejima, Susumu Ohno, Nao Sugahara, Yasusato Oyama, Fumitaka Kawakita, Masao |
author_facet | Sakaguchi, Masayoshi Osaku, Kanae Maejima, Susumu Ohno, Nao Sugahara, Yasusato Oyama, Fumitaka Kawakita, Masao |
author_sort | Sakaguchi, Masayoshi |
collection | PubMed |
description | The proteinase K subfamily enzymes, thermophilic Aqualysin I (AQN) from Thermus aquaticus YT-1 and psychrophilic serine protease (VPR) from Vibrio sp. PA-44, have six and seven salt bridges, respectively. To understand the possible significance of salt bridges in the thermal stability of AQN, we prepared mutant proteins in which amino acid residues participating in salt bridges common to proteinase K subfamily members and intrinsic to AQN were replaced to disrupt the bridges one at a time. Disruption of a salt bridge common to proteinase K subfamily enzymes in the D183N mutant resulted in a significant reduction in thermal stability, and a massive change in the content of the secondary structure was observed, even at 70°C, in the circular dichroism (CD) analysis. These results indicate that the common salt bridge Asp183-Arg12 is important in maintaining the conformation of proteinase K subfamily enzymes and suggest the importance of proximity between the regions around Asp183 and the N-terminal region around Arg12. Of the three mutants that lack an AQN intrinsic salt bridge, D212N was more prone to unfolding at 80°C than the wild-type enzyme. Similarly, D17N and E237Q were less thermostable than the wild-type enzyme, although this may be partially due to increased autolysis. The AQN intrinsic salt bridges appear to confer additional thermal stability to this enzyme. These findings will further our understanding of the factors involved in stabilizing protein structure. |
format | Online Article Text |
id | pubmed-4131155 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Springer |
record_format | MEDLINE/PubMed |
spelling | pubmed-41311552014-08-18 Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1 Sakaguchi, Masayoshi Osaku, Kanae Maejima, Susumu Ohno, Nao Sugahara, Yasusato Oyama, Fumitaka Kawakita, Masao AMB Express Original Article The proteinase K subfamily enzymes, thermophilic Aqualysin I (AQN) from Thermus aquaticus YT-1 and psychrophilic serine protease (VPR) from Vibrio sp. PA-44, have six and seven salt bridges, respectively. To understand the possible significance of salt bridges in the thermal stability of AQN, we prepared mutant proteins in which amino acid residues participating in salt bridges common to proteinase K subfamily members and intrinsic to AQN were replaced to disrupt the bridges one at a time. Disruption of a salt bridge common to proteinase K subfamily enzymes in the D183N mutant resulted in a significant reduction in thermal stability, and a massive change in the content of the secondary structure was observed, even at 70°C, in the circular dichroism (CD) analysis. These results indicate that the common salt bridge Asp183-Arg12 is important in maintaining the conformation of proteinase K subfamily enzymes and suggest the importance of proximity between the regions around Asp183 and the N-terminal region around Arg12. Of the three mutants that lack an AQN intrinsic salt bridge, D212N was more prone to unfolding at 80°C than the wild-type enzyme. Similarly, D17N and E237Q were less thermostable than the wild-type enzyme, although this may be partially due to increased autolysis. The AQN intrinsic salt bridges appear to confer additional thermal stability to this enzyme. These findings will further our understanding of the factors involved in stabilizing protein structure. Springer 2014-08-13 /pmc/articles/PMC4131155/ /pubmed/25136511 http://dx.doi.org/10.1186/s13568-014-0059-2 Text en Copyright © 2014 Sakaguchi et al.; licensee Springer http://creativecommons.org/licenses/by/4.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. |
spellingShingle | Original Article Sakaguchi, Masayoshi Osaku, Kanae Maejima, Susumu Ohno, Nao Sugahara, Yasusato Oyama, Fumitaka Kawakita, Masao Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1 |
title | Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1 |
title_full | Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1 |
title_fullStr | Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1 |
title_full_unstemmed | Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1 |
title_short | Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1 |
title_sort | highly conserved salt bridge stabilizes a proteinase k subfamily enzyme, aqualysin i, from thermus aquaticus yt-1 |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4131155/ https://www.ncbi.nlm.nih.gov/pubmed/25136511 http://dx.doi.org/10.1186/s13568-014-0059-2 |
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