Controlled Immobilization of Membrane Proteins to Surfaces for Fourier Transform Infrared Investigations

Per Rigler, Horst Vogel
2004
Article

Résumé

We show that it is possible to immobilize membrane proteins uniformly and reversibly as self-assembled (sub)monolayers on nitrilotriacetic acid-covered sensor surfaces via hexahistidine sequences present either in the protein or in lipid membranes. Fourier transform IR spectra of such self-assembled (sub)monolayers deliver important structural information of the membrane proteins and are suited to screen the function of cellular receptors. [on SciFinder (R)]

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https://infoscience.epfl.ch/record/81120?ln=fr

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Downscaling Fourier Transform Infrared Spectroscopy to the Micrometer and Nanogram Scale: Secondary Structure of Serotonin and Acetylcholine Receptors

Rudolf Hovius, Erwin Ilegems, Horst Pick, Per Rigler, Horst Vogel

High signal-to-noise Fourier transform IR (FTIR) spectra of the 5-hydroxytryptamine (serotonin) receptor (5-HT3R) and the nicotinic acetylcholine receptor (nAChR) were obtained by microscope FTIR spectroscopy using micrometer-sized, fully hydrated protein films. Because this novel procedure requires only nanogram quantities of membrane proteins, which is 4-5 orders of magnitude less than the amt. of protein typically used for conventional FTIR spectroscopy, it opens the possibility to access the structure and dynamics of many important mammalian receptor proteins. The secondary structure of detergent-solubilized 5-HT3R detd. by curve fitting of the amide I band yielded 36% a-helix, 33% b-strand, 15% b-turn, and 16% nonregular structures, which remained unchanged upon reconstitution in lipid membranes. From hydrogen-deuterium exchange, the secondary structure of the water-accessible part of 5-HT3R was detd. as 14% a-helix, 16% b-strand, 26% b-turn, and 14% nonregular structures. Interestingly, we found that both the overall and the water-accessible nAChR secondary structures were nearly identical to those of 5-HT3R, in agreement with predicted structures of this class of receptors. This is the first time that structural investigations were obtained for two closely related ligand-gated ion channels under strictly identical exptl. conditions. [on SciFinder (R)]

2003

Chargement

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The nicotinic acetylcholine receptor from Torpedo was immobilised in tethered membranes. Surface plasmon resonance was used to quantify the binding of ligands and antibodies to the receptor. The orientation and structural integrity of the surface-reconstituted receptor was probed using monoclonal antibodies, demonstrating that approximately 65% of the receptors present their ligand-binding site towards the lumen of the flow cell and that at least 85% of these receptors are structurally intact. The conformation of the receptor in tethered membranes was investigated with Fourier transform infrared spectroscopy and found to be practically identical to that of receptors reconstituted in lipid vesicles. The affinity of small receptor ligands was determined in a competition assay against a monoclonal antibody directed against the ligand-binding site which yielded dissociation constants in agreement with radioligand binding assays. The presented method for the functional immobilisation of the nicotinic acetylcholine receptor in tethered membranes might be generally applicable to other membrane proteins.

Elsevier2000

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In a recent paper, a new class of synthetic lipids, which self-assemble to form monolayers on gold surfaces, was introduced. The addn. of a monolayer of phospholipids to these layers results in robust supported lipid bilayers designed to reconstitute transmembrane proteins. This paper presents a detailed characterization of the monolayers and bilayers formed by these lipids and assesses their ability for the incorporation of membrane-spanning proteins. Film balance methods, electron microscopy, surface plasmon resonance, Fourier transform IR measurements and Raman spectroscopy show the feasibility of this concept. However, further modifications to improve the thiolipids are necessary. A new strategy for the formation of laterally structured supported lipid bilayers is discussed and initial results are presented. [on SciFinder (R)]

1996

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