The browser you are using is not supported by this website. All versions of Internet Explorer are no longer supported, either by us or Microsoft (read more here: https://www.microsoft.com/en-us/microsoft-365/windows/end-of-ie-support).

Please use a modern browser to fully experience our website, such as the newest versions of Edge, Chrome, Firefox or Safari etc.

Picture of Swati Aggarwal

Swati Aggarwal

Researcher

Picture of Swati Aggarwal

A protocol for production of perdeuterated OmpF porin for neutron crystallography

Author

  • Swati Aggarwal
  • Claes von Wachenfeldt
  • Suzanne Zoë Fisher
  • Esko Oksanen

Summary, in English

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.

Department/s

  • Molecular Cell Biology
  • Department of Biology

Publishing year

2021-12

Language

English

Publication/Series

Protein Expression and Purification

Volume

188

Document type

Journal article

Publisher

Academic Press

Topic

  • Structural Biology

Keywords

  • Deuteration
  • Membrane proteins
  • Neutron crystallography
  • Neutron scattering
  • OmpF porin

Status

Published

ISBN/ISSN/Other

  • ISSN: 1046-5928