Cryo-electron microscopic and X-ray crystallographic analysis of the light-driven proton pump proteorhodopsin reveals a pentameric assembly.

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 and Fotiadis, Dimitrios José

Subjects:

500 Science > 570 Life sciences; biology
600 Technology > 610 Medicine & health

ISSN:

2590-1524

Publisher:

Elsevier

Language:

English

Submitter:

Barbara Järmann-Bangerter

Date Deposited:

01 Sep 2020 11:05

Last Modified:

06 Sep 2020 03:05

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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