A protocol for production of perdeuterated OmpF porin for neutron crystallography
(2021) In Protein Expression and Purification 188.- Abstract
Hydrogen atoms are at the limit of visibility in X-ray structures even at high resolution. Neutron macromolecular crystallography (NMX) is an unambiguous method to locate hydrogens and study the significance of hydrogen bonding interactions in biological systems. Since NMX requires very large crystals, very few neutron structures of proteins have been determined yet. In addition, the most common hydrogen isotope 1H gives rise to significant background due to its large incoherent scattering cross-section. Therefore, it is advantageous to substitute as many hydrogens as possible with the heavier isotope 2H (deuterium) to reduce the sample volume requirement. While the solvent exchangeable hydrogens can be substituted... (More)
Hydrogen atoms are at the limit of visibility in X-ray structures even at high resolution. Neutron macromolecular crystallography (NMX) is an unambiguous method to locate hydrogens and study the significance of hydrogen bonding interactions in biological systems. Since NMX requires very large crystals, very few neutron structures of proteins have been determined yet. In addition, the most common hydrogen isotope 1H gives rise to significant background due to its large incoherent scattering cross-section. Therefore, it is advantageous to substitute as many hydrogens as possible with the heavier isotope 2H (deuterium) to reduce the sample volume requirement. While the solvent exchangeable hydrogens can be substituted by dissolving the protein in heavy water, complete deuterium labelling – perdeuteration – requires the protein to be expressed in heavy water with a deuterated carbon source. In this work, we developed an optimized method for large scale production of deuterium-labelled bacterial outer membrane protein F (OmpF) for NMX. OmpF was produced using deuterated media with different carbon sources. Mass spectrometry verified the integrity and level of deuteration of purified OmpF. Perdeuterated OmpF crystals diffracted X-rays to a resolution of 1.9 Å. This work lays the foundation for structural studies of membrane protein by neutron diffraction in future.
(Less)
- author
- Aggarwal, Swati LU ; Wachenfeldt, Claes von LU ; Fisher, Suzanne Zoë LU and Oksanen, Esko LU
- organization
- publishing date
- 2021-12
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Deuteration, Membrane proteins, Neutron crystallography, Neutron scattering, OmpF porin
- in
- Protein Expression and Purification
- volume
- 188
- article number
- 105954
- publisher
- Academic Press
- external identifiers
-
- scopus:85113681582
- pmid:34416360
- ISSN
- 1046-5928
- DOI
- 10.1016/j.pep.2021.105954
- language
- English
- LU publication?
- yes
- id
- 04f4dd76-e74d-4064-bd6c-293d1d23aa6f
- date added to LUP
- 2021-09-17 13:11:52
- date last changed
- 2024-06-15 16:30:37
@article{04f4dd76-e74d-4064-bd6c-293d1d23aa6f,
abstract = {{<p>Hydrogen atoms are at the limit of visibility in X-ray structures even at high resolution. Neutron macromolecular crystallography (NMX) is an unambiguous method to locate hydrogens and study the significance of hydrogen bonding interactions in biological systems. Since NMX requires very large crystals, very few neutron structures of proteins have been determined yet. In addition, the most common hydrogen isotope <sup>1</sup>H gives rise to significant background due to its large incoherent scattering cross-section. Therefore, it is advantageous to substitute as many hydrogens as possible with the heavier isotope <sup>2</sup>H (deuterium) to reduce the sample volume requirement. While the solvent exchangeable hydrogens can be substituted by dissolving the protein in heavy water, complete deuterium labelling – perdeuteration – requires the protein to be expressed in heavy water with a deuterated carbon source. In this work, we developed an optimized method for large scale production of deuterium-labelled bacterial outer membrane protein F (OmpF) for NMX. OmpF was produced using deuterated media with different carbon sources. Mass spectrometry verified the integrity and level of deuteration of purified OmpF. Perdeuterated OmpF crystals diffracted X-rays to a resolution of 1.9 Å. This work lays the foundation for structural studies of membrane protein by neutron diffraction in future.</p>}},
author = {{Aggarwal, Swati and Wachenfeldt, Claes von and Fisher, Suzanne Zoë and Oksanen, Esko}},
issn = {{1046-5928}},
keywords = {{Deuteration; Membrane proteins; Neutron crystallography; Neutron scattering; OmpF porin}},
language = {{eng}},
publisher = {{Academic Press}},
series = {{Protein Expression and Purification}},
title = {{A protocol for production of perdeuterated OmpF porin for neutron crystallography}},
url = {{http://dx.doi.org/10.1016/j.pep.2021.105954}},
doi = {{10.1016/j.pep.2021.105954}},
volume = {{188}},
year = {{2021}},
}