Structural Determinants of Cold Adaptation and Stability in a Large Protein
peer reviewed The heat-labile alpha-amylase from an antarctic bacterium is the largest known protein that unfolds reversibly according to a two-state transition as shown by differential scanning calorimetry. Mutants of this enzyme were produced, carrying additional weak interactions found in thermos...
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ftorbi:oai:orbi.ulg.ac.be:2268/16026 2024-04-21T07:50:09+00:00 Structural Determinants of Cold Adaptation and Stability in a Large Protein D'Amico, Salvino Gerday, Charles Feller, Georges 2001-07-13 https://orbi.uliege.be/handle/2268/16026 https://orbi.uliege.be/bitstream/2268/16026/1/JBC_2001_MutAHA.pdf https://doi.org/10.1074/jbc.M102741200 en eng American Society for Biochemistry and Molecular Biology urn:issn:0021-9258 urn:issn:1083-351X https://orbi.uliege.be/handle/2268/16026 info:hdl:2268/16026 https://orbi.uliege.be/bitstream/2268/16026/1/JBC_2001_MutAHA.pdf doi:10.1074/jbc.M102741200 scopus-id:2-s2.0-0035854688 info:pmid:11325973 open access http://purl.org/coar/access_right/c_abf2 info:eu-repo/semantics/openAccess Journal of Biological Chemistry, 276 (28), 25791-6 (2001-07-13) Life sciences Biochemistry biophysics & molecular biology Sciences du vivant Biochimie biophysique & biologie moléculaire journal article http://purl.org/coar/resource_type/c_6501 info:eu-repo/semantics/article peer reviewed 2001 ftorbi https://doi.org/10.1074/jbc.M102741200 2024-03-27T14:52:24Z peer reviewed The heat-labile alpha-amylase from an antarctic bacterium is the largest known protein that unfolds reversibly according to a two-state transition as shown by differential scanning calorimetry. Mutants of this enzyme were produced, carrying additional weak interactions found in thermostable alpha-amylases. It is shown that single amino acid side chain substitutions can significantly modify the melting point T(m), the calorimetric enthalpy Delta H(cal), the cooperativity and reversibility of unfolding, the thermal inactivation rate constant, and the kinetic parameters k(cat) and K(m). The correlation between thermal inactivation and unfolding reversibility displayed by the mutants also shows that stabilizing interactions increase the frequency of side reactions during refolding, leading to intramolecular mismatches or aggregations typical of large proteins. Although all mutations were located far from the active site, their overall trend is to decrease both k(cat) and K(m) by rigidifying the molecule and to protect mutants against thermal inactivation. The effects of these mutations indicate that the cold-adapted alpha-amylase has lost a large number of weak interactions during evolution to reach the required conformational plasticity for catalysis at low temperatures, thereby producing an enzyme close to the lowest stability allowing maintenance of the native conformation. Article in Journal/Newspaper Antarc* Antarctic University of Liège: ORBi (Open Repository and Bibliography) Journal of Biological Chemistry 276 28 25791 25796 |
institution |
Open Polar |
collection |
University of Liège: ORBi (Open Repository and Bibliography) |
op_collection_id |
ftorbi |
language |
English |
topic |
Life sciences Biochemistry biophysics & molecular biology Sciences du vivant Biochimie biophysique & biologie moléculaire |
spellingShingle |
Life sciences Biochemistry biophysics & molecular biology Sciences du vivant Biochimie biophysique & biologie moléculaire D'Amico, Salvino Gerday, Charles Feller, Georges Structural Determinants of Cold Adaptation and Stability in a Large Protein |
topic_facet |
Life sciences Biochemistry biophysics & molecular biology Sciences du vivant Biochimie biophysique & biologie moléculaire |
description |
peer reviewed The heat-labile alpha-amylase from an antarctic bacterium is the largest known protein that unfolds reversibly according to a two-state transition as shown by differential scanning calorimetry. Mutants of this enzyme were produced, carrying additional weak interactions found in thermostable alpha-amylases. It is shown that single amino acid side chain substitutions can significantly modify the melting point T(m), the calorimetric enthalpy Delta H(cal), the cooperativity and reversibility of unfolding, the thermal inactivation rate constant, and the kinetic parameters k(cat) and K(m). The correlation between thermal inactivation and unfolding reversibility displayed by the mutants also shows that stabilizing interactions increase the frequency of side reactions during refolding, leading to intramolecular mismatches or aggregations typical of large proteins. Although all mutations were located far from the active site, their overall trend is to decrease both k(cat) and K(m) by rigidifying the molecule and to protect mutants against thermal inactivation. The effects of these mutations indicate that the cold-adapted alpha-amylase has lost a large number of weak interactions during evolution to reach the required conformational plasticity for catalysis at low temperatures, thereby producing an enzyme close to the lowest stability allowing maintenance of the native conformation. |
format |
Article in Journal/Newspaper |
author |
D'Amico, Salvino Gerday, Charles Feller, Georges |
author_facet |
D'Amico, Salvino Gerday, Charles Feller, Georges |
author_sort |
D'Amico, Salvino |
title |
Structural Determinants of Cold Adaptation and Stability in a Large Protein |
title_short |
Structural Determinants of Cold Adaptation and Stability in a Large Protein |
title_full |
Structural Determinants of Cold Adaptation and Stability in a Large Protein |
title_fullStr |
Structural Determinants of Cold Adaptation and Stability in a Large Protein |
title_full_unstemmed |
Structural Determinants of Cold Adaptation and Stability in a Large Protein |
title_sort |
structural determinants of cold adaptation and stability in a large protein |
publisher |
American Society for Biochemistry and Molecular Biology |
publishDate |
2001 |
url |
https://orbi.uliege.be/handle/2268/16026 https://orbi.uliege.be/bitstream/2268/16026/1/JBC_2001_MutAHA.pdf https://doi.org/10.1074/jbc.M102741200 |
genre |
Antarc* Antarctic |
genre_facet |
Antarc* Antarctic |
op_source |
Journal of Biological Chemistry, 276 (28), 25791-6 (2001-07-13) |
op_relation |
urn:issn:0021-9258 urn:issn:1083-351X https://orbi.uliege.be/handle/2268/16026 info:hdl:2268/16026 https://orbi.uliege.be/bitstream/2268/16026/1/JBC_2001_MutAHA.pdf doi:10.1074/jbc.M102741200 scopus-id:2-s2.0-0035854688 info:pmid:11325973 |
op_rights |
open access http://purl.org/coar/access_right/c_abf2 info:eu-repo/semantics/openAccess |
op_doi |
https://doi.org/10.1074/jbc.M102741200 |
container_title |
Journal of Biological Chemistry |
container_volume |
276 |
container_issue |
28 |
container_start_page |
25791 |
op_container_end_page |
25796 |
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1796933988978262016 |