Structural and functional characterization of proteins as well as the design of targeted drugs heavily rely on recombinant protein expression and purification. The polyhistidine-tag (His-tag) is among the most prominent examples of affinity tags used for the isolation of recombinant proteins from their expression hosts. Short peptide tags are commonly considered not to interfere with the structure of the tagged protein and tag removal is frequently neglected. This study demonstrates the formation of higher-order oligomers based on the example of two His-tagged membrane proteins, the dimeric arginine-agmatine antiporter AdiC and the pentameric light-driven proton pump proteorhodopsin. Size exclusion chromatography revealed the formation of tetrameric AdiC and decameric as well as pentadecameric proteorhodopsin through specific interactions between their His-tags. In addition, single particle cryo-electron microscopy (cryo-EM) allowed structural insights into the three-dimensional arrangement of the higher-order oligomers and the underlying His-tag-mediated interactions. These results reinforce the importance of considering the length and removal of affinity purification tags and illustrate how neglect can lead to potential interference with downstream biophysical or biochemical characterization of the target protein.

The light-driven proton pump bacteriorhodopsin (BR) from the extreme halophilic archaeon Halobacterium salinarum is a retinal-binding protein, which forms highly ordered and thermally stable 2D crystals in native membranes (termed purple membranes). BR and purple membranes (PMs) have been and are still being intensively studied by numerous researchers from different scientific disciplines. Furthermore, PMs are being successfully used in new, emerging technologies such as bioelectronics and bionanotechnology. Most published studies used the wild-type form of BR, because of the intrinsic difficulty to produce genetically modified versions in purple membranes homologously. However, modification and engineering is crucial for studies in basic research and, in particular, to tailor BR for specific applications in applied sciences. We present an extensive and detailed protocol ranging from the genetic modification and cultivation of H. salinarum to the isolation, and biochemical, biophysical and functional characterization of BR and purple membranes. Pitfalls and problems of the homologous expression of BR versions in H. salinarum are discussed and possible solutions presented. The protocol is intended to facilitate the access to genetically modified BR versions for researchers of different scientific disciplines, thus increasing the application of this versatile biomaterial.

• Affinity tag-free isolation of proteorhodopsin (PR) by ion exchange and size exclusion chromatography. • Biochemical and computational analysis indicate a single, pentameric PR population. • Highly pure and homogeneous PR enables growth of 3D crystals. • X-ray crystallography and cryo-electron microscopy reveal pentameric assembly of PR.

Microbial ion-pumping rhodopsins (MRs) are extensively studied retinal-binding membrane proteins. However, their biogenesis, including oligomerisation and retinal incorporation, remains poorly understood. The bacterial green-light absorbing proton pump proteorhodopsin (GPR) has emerged as a model protein for MRs and is used here to address these open questions using cryo-electron microscopy (cryo-EM) and molecular dynamics (MD) simulations. Specifically, conflicting studies regarding GPR stoichiometry reported pentamer and hexamer mixtures without providing possible assembly mechanisms. We report the pentameric and hexameric cryo-EM structures of a GPR mutant, uncovering the role of the unprocessed N-terminal signal peptide in the assembly of hexameric GPR. Furthermore, certain proteorhodopsin-expressing bacteria lack retinal biosynthesis pathways, suggesting that they scavenge the cofactor from their environment. We shed light on this hypothesis by solving the cryo-EM structure of retinal-free proteoopsin, which together with mass spectrometry and MD simulations suggests that decanoate serves as a temporary placeholder for retinal in the chromophore binding pocket. Further MD simulations elucidate possible pathways for the exchange of decanoate and retinal, offering a mechanism for retinal scavenging. Collectively, our findings provide insights into the biogenesis of MRs, including their oligomeric assembly, variations in protomer stoichiometry and retinal incorporation through a potential cofactor scavenging mechanism.

Data underlying the figures in the publication “Cryo-EM structure and dynamics of the green-light absorbing proteorhodopsin”, published in Nat Commun, 2021, 12, 4107. https://doi.org/10.1038/s41467-021-24429-6 Table of contents: 1. Dataset; Excel file containing the numerical data for Figures S1A and 4C. 2. Methods; Word file containing the description of the methods. The cryo-EM map was deposited in the Electron Microscopy Data Bank (EMDB) under accession code EMD-11955 and the protein coordinates were deposited in the Protein Data Bank (PDB) with the accession code 7B03. EMDB: https://www.emdataresource.org/EMD-11955 PDB: https://www.rcsb.org/structure/7B03 (DOI: 10.2210/pdb7B03/pdb)