Controlled immobilization of membrane proteins to surfaces for fourier transform infrared investigations.

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 infrared 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.

Downscaling Fourier transform infrared spectroscopy to the micrometer and nanogram scale: secondary structure of serotonin and acetylcholine receptors.

High signal-to-noise Fourier transform infrared (FTIR) spectra of the 5-hydroxytryptamine (serotonin) receptor (5-HT(3)R) 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 amount 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-HT(3)R determined by curve fitting of the amide I band yielded 36% alpha-helix, 33% beta-strand, 15% beta-turn, and 16% nonregular structures, which remained unchanged upon reconstitution in lipid membranes.

Reversible immobilization of peptides: surface modification and in situ detection by attenuated total reflection FTIR spectroscopy.

A generic method is described for the reversible immobilization of polyhistidine-bearing polypeptides and proteins on attenuated total reflecting (ATR) sensor surfaces for the detection of biomolecular interactions by FTIR spectroscopy. Nitrilotriacetic acid (NTA) groups are covalently attached to self-assembled monolayers of either thioalkanes on gold films or mercaptosilanes on silicon dioxide films deposited on germanium internal reflection elements. Complex formation between Ni2+ ions and NTA groups activates the ATR sensor surface for the selective binding of polyhistidine sequences.

Stable self-assembly of a protein engineering scaffold on gold surfaces.

The outer membrane protein OmpF from Escherichia coli is a member of a large family of beta-barrel membrane proteins. Some, like OmpF, are pore-forming proteins whilse others are active transporters or enzymes. We have previously shown that the receptor-binding domain (R-domain) of the toxin colicin N binds with high affinity to OmpF reconstituted into tethered lipid bilayers on gold electrodes.

Self-assembly of the hydrophobin SC3 proceeds via two structural intermediates.

Hydrophobins self assemble into amphipathic films at hydrophobic-hydrophilic interfaces. These proteins are involved in a broad range of processes in fungal development. We have studied the conformational changes that accompany the self-assembly of the hydrophobin SC3 with polarization-modulation infrared reflection absorption spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy, and circular dichroism, and related them to changes in morphology as observed by electron microcopy.