Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze protein.
The highly homologous type III antifreeze protein (AFP) subfamily share the capability to inhibit ice growth at subzero temperatures. Extensive studies by X-ray crystallography have been conducted, mostly on AFPs from polar fishes. Although interactions between a defined flat ice-binding surface and...
Published in: | Acta Crystallographica Section F Structural Biology and Crystallization Communications |
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Online Access: | https://doi.org/10.1107/S1744309109008574 https://pubmed.ncbi.nlm.nih.gov/19342793 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2664773/ |
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ftpubmed:19342793 2024-06-09T07:48:14+00:00 Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze protein. Petit-Haertlein, Isabelle Blakeley, Matthew P Howard, Eduardo Hazemann, Isabelle Mitschler, Andre Haertlein, Michael Podjarny, Alberto 2009 Apr 01 https://doi.org/10.1107/S1744309109008574 https://pubmed.ncbi.nlm.nih.gov/19342793 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2664773/ eng eng International Union of Crystallography https://doi.org/10.1107/S1744309109008574 https://pubmed.ncbi.nlm.nih.gov/19342793 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2664773/ Acta Crystallogr Sect F Struct Biol Cryst Commun ISSN:1744-3091 Volume:65 Issue:Pt 4 Journal Article Research Support, Non-U.S. Gov't 2009 ftpubmed https://doi.org/10.1107/S1744309109008574 2024-05-16T16:03:00Z The highly homologous type III antifreeze protein (AFP) subfamily share the capability to inhibit ice growth at subzero temperatures. Extensive studies by X-ray crystallography have been conducted, mostly on AFPs from polar fishes. Although interactions between a defined flat ice-binding surface and a particular lattice plane of an ice crystal have now been identified, the fine structural features underlying the antifreeze mechanism still remain unclear owing to the intrinsic difficulty in identifying H atoms using X-ray diffraction data alone. Here, successful perdeuteration (i.e. complete deuteration) for neutron crystallographic studies of the North Atlantic ocean pout (Macrozoarces americanus) AFP in Escherichia coli high-density cell cultures is reported. The perdeuterated protein (AFP D) was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Well shaped perdeuterated AFP D crystals have been grown in D(2)O by the sitting-drop method. Preliminary neutron Laue diffraction at 293 K using LADI-III at ILL showed that with a few exposures of 24 h a very low background and clear small spots up to a resolution of 1.85 A were obtained using a ;radically small' perdeuterated AFP D crystal of dimensions 0.70 x 0.55 x 0.35 mm, corresponding to a volume of 0.13 mm(3). Article in Journal/Newspaper North Atlantic PubMed Central (PMC) Acta Crystallographica Section F Structural Biology and Crystallization Communications 65 4 406 409 |
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PubMed Central (PMC) |
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English |
description |
The highly homologous type III antifreeze protein (AFP) subfamily share the capability to inhibit ice growth at subzero temperatures. Extensive studies by X-ray crystallography have been conducted, mostly on AFPs from polar fishes. Although interactions between a defined flat ice-binding surface and a particular lattice plane of an ice crystal have now been identified, the fine structural features underlying the antifreeze mechanism still remain unclear owing to the intrinsic difficulty in identifying H atoms using X-ray diffraction data alone. Here, successful perdeuteration (i.e. complete deuteration) for neutron crystallographic studies of the North Atlantic ocean pout (Macrozoarces americanus) AFP in Escherichia coli high-density cell cultures is reported. The perdeuterated protein (AFP D) was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Well shaped perdeuterated AFP D crystals have been grown in D(2)O by the sitting-drop method. Preliminary neutron Laue diffraction at 293 K using LADI-III at ILL showed that with a few exposures of 24 h a very low background and clear small spots up to a resolution of 1.85 A were obtained using a ;radically small' perdeuterated AFP D crystal of dimensions 0.70 x 0.55 x 0.35 mm, corresponding to a volume of 0.13 mm(3). |
format |
Article in Journal/Newspaper |
author |
Petit-Haertlein, Isabelle Blakeley, Matthew P Howard, Eduardo Hazemann, Isabelle Mitschler, Andre Haertlein, Michael Podjarny, Alberto |
spellingShingle |
Petit-Haertlein, Isabelle Blakeley, Matthew P Howard, Eduardo Hazemann, Isabelle Mitschler, Andre Haertlein, Michael Podjarny, Alberto Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze protein. |
author_facet |
Petit-Haertlein, Isabelle Blakeley, Matthew P Howard, Eduardo Hazemann, Isabelle Mitschler, Andre Haertlein, Michael Podjarny, Alberto |
author_sort |
Petit-Haertlein, Isabelle |
title |
Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze protein. |
title_short |
Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze protein. |
title_full |
Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze protein. |
title_fullStr |
Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze protein. |
title_full_unstemmed |
Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze protein. |
title_sort |
perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type iii antifreeze protein. |
publisher |
International Union of Crystallography |
publishDate |
2009 |
url |
https://doi.org/10.1107/S1744309109008574 https://pubmed.ncbi.nlm.nih.gov/19342793 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2664773/ |
genre |
North Atlantic |
genre_facet |
North Atlantic |
op_source |
Acta Crystallogr Sect F Struct Biol Cryst Commun ISSN:1744-3091 Volume:65 Issue:Pt 4 |
op_relation |
https://doi.org/10.1107/S1744309109008574 https://pubmed.ncbi.nlm.nih.gov/19342793 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2664773/ |
op_doi |
https://doi.org/10.1107/S1744309109008574 |
container_title |
Acta Crystallographica Section F Structural Biology and Crystallization Communications |
container_volume |
65 |
container_issue |
4 |
container_start_page |
406 |
op_container_end_page |
409 |
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1801379872887537664 |