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. From hydrogen-deuterium exchange, the secondary structure of the water-accessible part of 5-HT(3)R was determined as 14% alpha-helix, 16% beta-strand, 26% beta-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-HT(3)R, 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 experimental conditions.

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. This approach not only allows a stable and reversible immobilization of histidine-tagged peptides (His-peptides) but also simultaneously allows the direct in situ quantification of surface-adsorbed molecules from their specific FTIR spectral bands. The surface concentrations of both NTA and His-peptide on silanized surfaces were determined to be 1.1 and 0.4 molecules nm-2, respectively, which means that the surface is densely covered. A comparison of experimental FTIR spectra with simulated spectra reveals a surface-enhancement effect of one order of magnitude for the gold surfaces. With the presented sensor surfaces, new ways are opened up to investigate, in situ and with high sensitivity and reproducibility, protein-ligand, protein-protein, protein-DNA interactions, and DNA hybridization by ATR-FTIR spectroscopy.

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. The binding can be measured by surface plasmon resonance (SPR) and ion channel blockage (impedance spectroscopy, IS). In this paper we report the use of a mutant OmpF-E183C in which a single cysteine had been introduced on a short periplasmic turn. OmpF-E183C binds directly to gold surfaces and creates high-density protein layers by self-assembly from detergent solution. When the gold surface is pretreated with beta-mercaptoethanol and thiolipids are added after the protein immobilisation step, the protein is shown, by Fourier transform infrared spectroscopy (FTIR), to retain its beta-rich structure. Furthermore, we could also measure R-domain binding by SPR and IS, confirming the functional reconstitution of a self-assembled membrane protein monolayer at the gold surface. Because these beta-barrel proteins are recognized protein engineering scaffolds, the method provides a generic method for the simple self-assembly of protein interfaces from aqueous solution.

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. Three states of SC3 have been spectroscopically identified previously as follows: the monomeric state, the alpha-helical state that is formed upon binding to a hydrophobic solid, and the beta-sheet state, which is formed at the air-water interface. Here, we show that the formation of the beta-sheet state of SC3 proceeds via two intermediates. The first intermediate has an infrared spectrum indistinguishable from that of the alpha-helical state of SC3. The second intermediate is rich in beta-sheet structure and has a featureless appearance under the electron microscope. The end state has the same secondary structure, but is characterized by the familiar 10-nm-wide rodlets.