SEIRA Spectroscopy on a Membrane Receptor Monolayer Using Lipoprotein Particles as Carriers (original) (raw)
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Biopolymers, 2006
We present a novel infrared method to investigate the functionality of a protein monolayer tethered to a metal substrate. The approach employs Surface Enhanced Infrared Absorption Spectroscopy (SEIRAS), which renders high surface sensitivity by enhancing the signal of the adsorbed protein by up to ∼2 orders of magnitude. We demonstrate that the electrochemically induced absorption changes of a cytochrome c monolayer can be observed with excellent signal‐to‐noise ratio when the protein is adhered to a modified gold surface. To probe membrane proteins, a concept is introduced for the oriented incorporation into solid supported lipid bilayers. Recombinant cytochrome c oxidase solubilized in detergent is immobilized on a chemically modified gold surface via the affinity of its histidine (His)‐tag to a nickel‐chelating nitro‐triacetic acid (NTA) surface. The protein monolayer is reconstituted into the lipid environment by detergent removal. Changing the orientation of the protein with re...
Disc Antenna Enhanced Infrared Spectroscopy: From Self-Assembled Monolayers to Membrane Proteins
ACS Sensors
Plasmonic surfaces have emerged as a powerful platform for biomolecular sensing applications and can be designed to optimize the plasmonic resonance for probing molecular vibrations at utmost sensitivity. Here, we present a facile procedure to generate metallic micro disc antenna arrays that are employed in surface-enhanced infrared absorption (SEIRA) spectroscopy of biomolecules. Transmission electron microscopy (TEM) grids are used as shadow mask deployed during physical vapor deposition of gold. The resulting disc-shaped antennas exhibit enhancement factors of the vibrational bands of 4×10 4 giving rise to a detection limit < 1 femtomol (10-15 mol) of molecules. Surface-bound monolayers of 4-mercaptobenzoic acid show polyelectrolyte behavior when titrated with cations in the aqueous medium. Conformational rigidity of the self-assembled monolayer is validated by density functional theory calculations. The membrane protein sensory rhodopsin II is tethered to the disc antenna arrays and is fully functional as inferred from the light-induced SEIRA difference spectra. As an advance to previous studies, the accessible frequency range is improved and extended into the fingerprint region.
Journal of Molecular Biology, 2008
Human apolipoprotein A-I (apo A-I) and its engineered constructs form discoidal lipid bilayers upon interaction with lipids in vitro. We now report the cloning, expression, and purification of apo A-I derived from zebrafish (Danio rerio), which combines with phospholipids to form similar discoidal bilayers and may prove to be superior to human apo A-I constructs for rapid reconstitution of seven-transmembrane helix receptors into nanoscale apolipoprotein bound bilayers (NABBs). We characterized NABBs by gelfiltration chromatography, native polyacrylamide gradient gel electrophoresis, UV-visible photobleaching difference spectroscopy, and fluorescence spectroscopy. We used electron microscopy to determine the stoichiometry and orientation of rhodopsin (rho)-containing NABBs prepared under various conditions and correlated stability and signaling efficiency of rho in NABBs with either one or two receptors. We discovered that the specific activity of G protein coupling for single rhos sequestered in individual NABBs was nearly identical with that of two rhos per NABB under conditions where stoichiometry and orientation could be inferred by electron microscopy imaging. Thermal stability of rho in NABBs was superior to that of rho in various commonly used detergents. We conclude that the NABB system using engineered zebrafish apo A-I is a native-like membrane mimetic system for G-protein-coupled receptors and discuss strategies for rapid incorporation of expressed membrane proteins into NABBs.
Spectroscopy, 2002
Planar lipid bilayers on sensor chip surfaces have become useful tools to study membrane bound processes by surface plasmon resonance spectroscopy. We immobilized phospholipids on sensor chips by different approaches. First, a self-assembled monolayer of octadecylmercaptan was formed on a blank gold surface and subsequent addition of phospholipids led to formation of a heterobilayer. Second, a self-assembled monolayer of mercaptoundecanoic acid was formed on a gold surface, the carboxy groups of mercaptoundecanoic acid were activated and covalently linked to phosphatidylethanolamine. Addition of phospholipids then led to a bilayer with phosphatidylethanolamine as the lower leaflet. Third, a hydrophobic sensor chip (L1, BIAcore) was used as a binding matrix for phospholipids. These lipid surfaces were tested, whether they are suitable to study proteinamembrane interactions. As biological test system we used the Ca2+-myristoyl-switch of the neuronal Ca2+-binding protein recoverin. All...
Peptide and protein binding to lipid monolayers studied by FT-IRRA spectroscopy
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2013
Lipid monolayers at the air-water interface represent half of a lipid bilayer and are therefore suitable model systems for studying the binding of peripheral proteins and polypeptides as well as proteins containing hydrophobic membrane anchors to membrane interfaces. Infrared reflection-absorption spectroscopy (IRRAS) of these monolayer films at the air-water interface provides information on the state of the lipid monolayers as well as on the conformational and orientational order of the film constituents. We will review shortly the experimental setup and the possibilities for obtaining structural information before several applications of the method to lipid-protein monolayers will be described. We will focus on examples where the analysis of the protein and peptide bands for pure monolayers of these compounds are combined with experiments where the same compounds are bound to lipid monolayers. Combination of these experiments leads to detailed information about the conformational properties and the orientation of the molecules at the air-water interface in contrast to being bound to the lipid-water interface. This article is part of a Special Issue entitled: FTIR in membrane proteins and peptide studies.
Polarization-dependent fluorescence of proteins bound to nanopore-confined lipid bilayers
The Journal of Chemical Physics, 2008
Lipid bilayers are essential structural component of biological membranes of all the living species: from viruses and bacteria to plants and humans. Biophysical and biochemical properties of such membranes are important for understanding physical mechanisms responsible for drug targeting. Binding events between proteins and the membrane may be ascertained by introducing fluorescence markers ͑chromophores͒ to the proteins. Here we describe a novel biosensing platform designed to enhance signals of these fluorescence markers. Nanoporous aluminum oxide membranes with and without gold ͑Au͒ surface coating have been employed for optical detection of bound conjugated streptavidin to biotinylated lipid bilayers-a model system that mimics protein docking to the membrane surface. Unexpectedly, it was found that fluorescence signals from such structures vary when pumped with E-polarized and H-polarized incident optical beams. The origin of the observed polarization-dependent effects and the implications for enhanced fluorescence detection in a biochip format are being discussed.
Biophysical Journal, 2005
Flash photolysis studies have shown that the membrane lipid environment strongly influences the ability of rhodopsin to form the key metarhodopsin II intermediate. Here we have used plasmon-waveguide resonance (PWR) spectroscopy, an optical method sensitive to both mass and conformation, to probe the effects of lipid composition on conformational changes of rhodopsin induced by light and due to binding and activation of transducin (G t ). Octylglucoside-solubilized rhodopsin was incorporated by detergent dilution into solid-supported bilayers composed either of egg phosphatidylcholine or various mixtures of a nonlamellar-forming lipid (dioleoylphosphatidylethanolamine; DOPE) together with a lamellar-forming lipid (dioleoylphosphatidylcholine; DOPC). Light-induced proteolipid conformational changes as a function of pH correlated well with previous flash photolysis studies, indicating that the PWR spectral shifts monitored metarhodopsin II formation. The magnitude of these effects, and hence the extent of the conformational transition, was found to be proportional to the DOPE content. Our data are consistent with previous suggestions that lipids having a negative spontaneous curvature favor elongation of rhodopsin during the activation process. In addition, measurements of the G t /rhodopsin interaction in a DOPC/DOPE (25:75) bilayer at pH 5 demonstrated that light activation increased the affinity for G t from 64 nM to 0.7 nM, whereas G t affinity for dark-adapted rhodopsin was unchanged. By contrast, in DOPC bilayers the affinity of G t for light-activated rhodopsin was only 18 nM at pH 5. Moreover exchange of GDP for GTPgS was also monitored by PWR spectroscopy. Only the light-activated receptor was able to induce this exchange which was unaffected by DOPE incorporation. These findings demonstrate that nonbilayer-forming lipids can alter functionally linked conformational changes of G-protein-coupled receptors in membranes, as well as their interactions with downstream effector proteins.
Nano-Chemical Infrared Imaging of Membrane Proteins in Lipid Bilayers
Journal of the American Chemical Society, 2013
The spectroscopic characterization of biomolecular structures requires nanometer spatial resolution and chemical specificity. We perform full spatio-spectral imaging of dried purple membrane patches purified from Halobacterium salinarum with infrared vibrational scattering-type scanning near-field optical microscopy (s-SNOM). Using near-field spectral phase contrast based on the Amide I resonance of the protein backbone, we identify the protein distribution with 20 nm spatial resolution and few-protein sensitivity. This demonstrates the general applicability of s-SNOM vibrational nanospectroscopy, with potential extension to a wide range of biomolecular systems.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2015
Membrane proteins (MPs) are prevalent drug discovery targets involved in many cell processes. Despite their high potential as drug targets, the study of MPs has been hindered by limitations in expression, purification and stabilization in order to acquire thermodynamic and kinetic parameters of small molecules binding. These bottlenecks are grounded on the mandatory use of detergents to isolate and extract MPs from the cell plasma membrane and the coexistence of multiple conformations, which reflects biochemical versatility and intrinsic instability of MPs. In this work ,we set out to define a new strategy to enable surface plasmon resonance (SPR) measurements on a thermostabilized and truncated version of the human adenosine (A 2A ) G-protein-coupled receptor (GPCR) inserted in a lipid bilayer nanodisc in a label-and detergent-free manner by using a combination of affinity tags and GFP-based fluorescence techniques. We were able to detect and characterize small molecules binding kinetics on a GPCR fully embedded in a lipid environment. By providing a comparison between different binding assays in membranes, nanodiscs and detergent micelles, we show that nanodiscs can be used for small molecule binding studies by SPR to enhance the MP stability and to trigger a more native-like behaviour when compared to kinetics on A 2A receptors isolated in detergent. This work provides thus a new methodology in drug discovery to characterize the binding kinetics of small molecule ligands for MPs targets in a lipid environment.