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Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

Published on: June 27, 2014

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

Stephan Hirschi¹, David Kalbermatter¹, Zöhre Ucurum¹

¹Institute of Biochemistry and Molecular Medicine, University of Bern, Bühlstrasse 28, 3012 Bern, Switzerland.

Journal of Structural Biology: X

|July 11, 2020

Abstract:

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.

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

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