Hirschi, Stephan; Kalbermatter, David; Ucurum, Zöhre; Fotiadis, Dimitrios (2020). Cryo-electron microscopic and X-ray crystallographic analysis of the light-driven proton pump proteorhodopsin reveals a pentameric assembly. Journal of structural biology: X, 4(100024), p. 100024. Elsevier 10.1016/j.yjsbx.2020.100024
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The green-light absorbing proteorhodopsin (GPR) is the prototype of bacterial light-driven proton pumps. It has been the focus of continuous research since its discovery 20 years ago and has sparked the development and application of various biophysical techniques. However, a certain controversy and ambiguity about the oligomeric assembly of GPR still remains. We present here the first tag-free purification of pentameric GPR. The combination of ion exchange and size exclusion chromatography yields homogeneous and highly pure untagged pentamers from GPR overexpressing Escherichia coli. The presented purification procedure provides native-like protein and excludes the need for affinity purification tags. Importantly, three-dimensional protein crystals of GPR were successfully grown and analyzed by X-ray crystallography. These results together with data from single particle cryo-electron microscopy provide direct evidence for the pentameric stoichiometry of purified GPR.
Item Type: |
Journal Article (Original Article) |
|---|---|
Division/Institute: |
04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine |
UniBE Contributor: |
Hirschi, Stephan, Kalbermatter, David, Ucurum Fotiadis, Zöhre, Fotiadis, Dimitrios José |
Subjects: |
500 Science > 570 Life sciences; biology 600 Technology > 610 Medicine & health |
ISSN: |
2590-1524 |
Publisher: |
Elsevier |
Language: |
English |
Submitter: |
Barbara Franziska Järmann-Bangerter |
Date Deposited: |
01 Sep 2020 11:05 |
Last Modified: |
05 Dec 2022 15:40 |
Publisher DOI: |
10.1016/j.yjsbx.2020.100024 |
PubMed ID: |
32647827 |
Uncontrolled Keywords: |
BN-PAGE, blue native-polyacrylamide gel electrophoresis BPR, blue-light absorbing proteorhodopsin BR, bacteriorhodopsin Cryo-electron microscopy GPR, green-light absorbing proteorhodopsin IEC, ion exchange chromatography MW, molecular weight Membrane protein Protein chromatography Proteorhodopsin Proton pump SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis SEC, size exclusion chromatography X-ray crystallography cryo-EM, cryo-electron microscopy |
BORIS DOI: |
10.7892/boris.146125 |
URI: |
https://boris.unibe.ch/id/eprint/146125 |
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