Visualizing Specific Cross-Protomer Interactions in the Homo-Oligomeric Membrane Protein Proteorhodopsin by Dynamic-Nuclear-Polarization-Enhanced Solid-State NMR

Abstract

Membrane proteins often form oligomeric complexes within the lipid bilayer, but factors controlling their assembly are hard to predict and experimentally difficult to determine. An understanding of protein-protein interactions within the lipid bilayer is however required in order to elucidate the role of oligomerization for their functional mechanism and stabilization. Here, we demonstrate for the pentameric, heptahelical membrane protein green proteorhodopsin that solid-state NMR could identify specific interactions at the protomer interfaces, if the sensitivity is enhanced by dynamic nuclear polarization. For this purpose, differently labeled protomers have been assembled into the full pentamer complex embedded within the lipid bilayer. We show for this proof of concept that one specific salt bridge determines the formation of pentamers or hexamers. Data are supported by laser-induced liquid bead ion desorption mass spectrometry and by blue native polyacrylamide gel electrophoresis analysis. The presented approach is universally applicable and opens the door toward analyzing membrane protein interactions within homo-oligomers directly in the membrane.

Dynamic Nuclear Polarization Signal Enhancement with High-Affinity Biradical Tags

“…4 One particularly robust mechanism, the cross effect, 5–8 uses nitroxide biradicals such as TOTAPOL cosolubilized with a system of interest, 9–11 with measurements performed near 100 K. DNP experiments with biradical polarizing agents have enabled studies of amyloidogenic peptides, bacteriorhodopsin, acetylcholine receptors, 12–15 and many other systems. 16–19 …”

New NMR tools for protein structure and function: Spin tags for dynamic nuclear polarization solid state NMR

“…DNP has been successfully applied to a number of biological systems, including rhodopsin membrane proteins [17–19], the Pf1 bacteriophage [20], transporter-bound peptides [21] and amyloid fibrils [22–24]. This has allowed observation of otherwise invisible NMR signals [18,20,25] from dilute components such as DNA in the Pf1 bacteriophage or peptides bound to membrane transporters [21,26] (see Fig.…”

Supramolecular Organization and Functional Implications of K+ Channel Clusters in Membranes

“…We overcame this issue with the DNP technique, which can provide sensitivity gains of two orders of magnitude, and thereby enables otherwise infeasible ssNMR experiments with lowly concentrated membrane proteins (for examples, see Refs. 9b, 14). We prepared vesicles with mixed 15 N/ 13 C‐labeled channels and ran experiments using a 263 GHz/400 MHz DNP/ssNMR setup at 100 K (see also the Supporting Information).…”