SEIRA Spectroscopy on a Membrane Receptor Monolayer Using Lipoprotein Particles as Carriers

Abstract

Surface-enhanced infrared absorption (SEIRA) difference spectroscopy can probe reactions in a protein monolayer tethered to a nanostructured gold surface. SEIRA studies of membrane proteins, however, remain challenging due to sample stability, effects of the metal surface on function, and the need for a membrane-mimicking environment. Here we demonstrate and characterize a model system for membrane receptor investigations using SEIRA spectroscopy. The system employs nanoscale apolipoprotein bound bilayer (NABB) particles, similar to discoidal high-density lipoprotein particles, as soluble carriers for the G-protein-coupled receptor rhodopsin. The His-tag of the engineered apolipoprotein allows for selective binding of the NABBs to a Ni-NTA modified surface, while the lipid environment of the particle ensures stability and protection of the embedded receptor. Using SEIRA spectroscopy, we followed specific binding of rhodopsin-loaded NABB particles to the surface and formation of a membrane protein monolayer. Functionality of the photoreceptor in the immobilized NABBs was probed by SEIRA difference spectroscopy confirming protein conformational changes associated with photoactivation. Orientation of the immobilized NABB particles was assessed by comparing SEIRA data with polarized attenuated total reflection-Fourier-transform infrared spectroscopy. Thus, SEIRA difference spectroscopy supported by the NABB technology provides a promising approach for further functional studies of transmembrane receptors.

Green Proteorhodopsin Reconstituted into Nanoscale Phospholipid Bilayers (Nanodiscs) as Photoactive Monomers

“…More sophisticated methods are available for studying the biochemical properties of various membrane proteins by different analytical methods. 25,36,37,40,82,83 …”

“…, the disc diameter) has allowed researchers to isolate the monomeric and multimeric protein conformations for proteins such as bacteriorhodopsin. 32–34 Lastly, these structures are being used in a wide variety of experimental settings that include NMR, 35 surface plasmon resonance, 36 as well as UV-visible, 29 infra-red 37 and fluorescence 28,38 spectroscopy. Nanodiscs thusly offer a versatile tool to deconstruct the factors that control the PR photocycle.…”

Surface-enhanced infrared absorption spectroscopy (SEIRAS) to probe monolayers of membrane proteins

“…Protein immobilization based on His-tag technology is highly versatile. However, the binding strength of the His-tag to monovalent NTA is just moderate (K D in the order of 10 -5 -10 -6 M) [34].…”

“…Cytochrome c was added to the solution and binding was monitored by SEIRAS for both CcO His-I and His-II. Although nanodiscs are a promising platform to study membrane proteins, only Vogel and coworkers reported a SEIRAS application up to now [34]. They incorporated the G-protein-coupled receptor (GPCR) rhodopsin into nanodisc particles.…”

Anisotropic metal growth on phospholipid nanodiscs via lipid bilayer expansion

“…Despite the confinement of the phospholipid bilayer by the MSP, NDs provide a native-like environment for membrane proteins which maintain their native structure and function 15 16 . Therefore, phospholipid NDs represent promising systems for the structural investigation of membrane proteins and their interaction with drug molecules by a variety of spectroscopic techniques including Raman scattering 17 and surface-enhanced infrared spectroscopy (SEIRS) 18 . Furthermore, NDs can also be used as building blocks for the self-assembly of larger hierarchical nanostructures.…”