Incorporation of the nicotinic acetylcholine receptor into planar multilamellar films: characterization by fluorescence and Fourier transform infrared difference spectroscopy.

A method for preparing thin, planar films of nicotinic acetylcholine receptor (nAChR) membranes that retain the ability to undergo the resting to desensitized state transition and that are suitable for spectroscopic studies has been developed. Native, alkaline-extracted nAChR membranes from Torpedo are dried under nitrogen on either a plastic microscope coverslip or a germanium internal reflection element (IRE) and then equilibrated with buffer. The drying procedure has no effect on the functional state of the nAChR as judged by a fluorescence assay using the probe ethidium bromide. The times required for an acetylcholine analogue (carbamylcholine), a local anesthetic (dibucaine), and a fluorescent probe (ethidium bromide) to penetrate films of varying degrees of thickness, interact with the receptor, and then to be washed from the films have been established. Under these conditions, the nAChR films can be repetitively cycled between the resting and desensitized states. Both fluorescence and infrared spectroscopy show that the films adhere strongly to either support even with buffer flowing continuously past the film surface. Fourier transform infrared difference spectra calculated from spectra recorded in the presence and absence of carbamylcholine show small, reproducible bands which reflect changes in nAChR structure upon desensitization.     

Effect of membrane lipid composition on the conformational equilibria of the nicotinic acetylcholine receptor

The effects of cholesterol (Chol) and an anionic lipid, dioleoylphosphatidic acid (DOPA) on the conformational equilibria of the nicotinic acetylcholine receptor (nAChR) have been investigated using Fourier transform infrared difference spectroscopy. The difference between spectra recorded in the presence and absence of agonist from the nAChR reconstituted into 3:1:1 egg phosphatidylcholine (EPC)/DOPA/Chol membranes exhibits positive and negative bands that serve as markers of the structural changes associated with the resting to desensitized conformational change. These markers are absent in similar difference spectra recorded from the nAChR reconstituted into EPC membranes lacking both Chol and DOPA, indicating that the nAChR cannot undergo conformational change in response to agonist binding. When low levels of either Chol or DOPA up to 25 mol % of the total lipid are included in the EPC membranes, the markers suggest the predominant stabilization of a conformation that is a structural intermediate between the resting and desensitized states. At higher levels of either Chol or DOPA, the nAChR is stabilized in a conformation that is capable of undergoing agonist-induced desensitization, although DOPA appears to be required for the nAChR to adopt a conformation fully equivalent to that found in native and 3:1:1 EPC/DOPA/Chol membranes. The ability of these two structurally diverse lipids, as well as others (Ryan, S. E., Demers, C. N., Chew, J. P., Baenziger, J. E. (1996) J. Biol. Chem. 271, 24590-24597), to modulate the functional state of the nAChR suggests that lipids act on the nAChR via an indirect effect on some physical property of the lipid bilayer. The data also suggest that anionic lipids are essential to stabilize a fully functional nAChR. We propose that membrane fluidity modulates the relative populations of nAChRs in the resting and desensitized states but that subtle structural changes in the presence of anionic lipids are essential for full activity.     

Fourier transform infrared spectroscopic characterization of a photolabile precursor of glutamate

Recently, it has been demonstrated that Fourier transform infrared spectroscopy (FTIR) detects conformational changes in the glutamate receptor ligand-binding domain that are associated with agonist binding. Combined with flash photolysis, this observation offers the prospect of following conformational changes at individual protein and agonist moieties in parallel and with high temporal resolution. Here, we demonstrate that gamma(alpha-carboxy-2-nitrobenzyl) glutamate (caged glutamate) does not interact with the protein, and that following photolysis with UV light the FTIR difference spectrum indicated changes in the protein tertiary and secondary interactions. These changes were similar to those observed for the protein upon addition of free glutamate. Thus, caged glutamate and its photolysis by-products are inert in this system, whereas the released glutamate exhibits full activity. Difference spectra of caged glutamate and of reaction analogs permitted identification of and correction for FTIR signals arising from the photolytic reaction and confirmed that its products are indeed glutamate and 2-nitrosophenyl glyoxalic acid.     

ATR-FTIR difference spectroscopy of the P(M) intermediate of bovine cytochrome c oxidase

Perfusion-induced attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to investigate changes induced in protein and cofactors of bovine cytochrome c oxidase when it was converted from the oxidised state to the catalytic P(M) intermediate. The transition was induced in a film of detergent-depleted 'fast' oxidase with a buffer containing CO and O(2). The extent of formation of the P(M) state was quantitated simultaneously by monitoring formation of its characteristic 607-nm band with a scanned visible beam reflected off the top surface of the prism. The P(M) minus O FTIR difference spectrum is distinctly different from the redox spectra reported to date and includes features that can be assigned to changes of haem a(3) and surrounding protein. Tentative assignments are made based on vibrational data of related proteins and model compounds.     

Conformational changes in bacteriorhodopsin studied by infrared attenuated total reflection.

We report on a new method based on Fourier transform infrared (FTIR)-difference spectroscopy for studying the conformational changes occurring during the photocycle of bacteriorhodopsin. Previous studies have been made by measuring the absorbance of an infrared (IR) beam transmitted through a thin hydrated purple membrane film. In contrast, the present study utilizes the technique of attenuated total reflection (ATR). Purple membrane is fixed on the surface of a germanium internal reflection crystal and immersed in a buffer whose pH and ionic composition can be varied. Measurements of the amide I and II absorbance with light polarized parallel and at 45 degrees to the crystal surface reveals that the membrane is highly oriented. An ATR-FTIR-difference spectrum of the light to dark (bR570 to bR548) transition is similar but not identical to the transmittance FTIR-difference spectrum. This disagreement between the two methods is shown to be due in the ATR case to the absorption of transition moments oriented predominantly out of the membrane plane. Raising the pH of La3+ substituted purple membrane films from 6.8 to 8.0 slows the M-decay rate sufficiently so that a bR570 to M412 difference spectrum can be obtained with steady state illumination at room temperature. A comparison of this difference spectrum with that obtained at -23 degrees C using the transmittance method reveals several changes that cannot be attributed to out-of-plane transition moments.(ABSTRACT TRUNCATED AT 250 WORDS)     

A nature of conformational changes of yeast tRNA(Phe). High hydrostatic pressure effects

We analysed conformational changes of yeast tRNA(Phe) induced by high hydrostatic pressure (HHP) measured by Fourier-transform infrared (FTIR) and fluorescence spectroscopies. High pressure influences RNA conformation without other cofactors, such as metal ions and salts. FTIR spectra of yeast tRNA(Phe) recorded at high hydrostatic pressure up to 13 kbar with and without magnesium ions showed a shift of the bands towards higher frequencies. That blue shift is due to an increase a higher energy of bonds as a result of shortening of hydrogen bonds followed by dehydration of tRNA. The fluorescence spectra of Y-base tRNA(Phe) at high pressure up to 3 kbar showed a decrease of the intensity band at 430 nm as a consequence of conformational rearrangement of the anticodon loop leading to exposure of Y-base side chain to the solution. We suggest that structural transition of nucleic acids is driven by the changes of water structure from tetrahedral to a cubic-like geometry induced by high pressure and, in consequence, due to economy of hydration.     

Attenuated total reflection IR spectroscopy as a tool to investigate the structure, orientation and tertiary structure changes in peptides and membrane proteins

During the last few years, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) has become one of the most powerful methods to determine the structure of biological materials and in particular of components of biological membranes, like proteins that cannot be studied by x-ray crystallography and NMR. ATR-FTIR requires a little amount of material (1-100 microg) and spectra are recorded in a matter of minutes. The environment of the molecules can be modulated so that their conformation can be studied as a function of temperature, pressure, pH, as well as in the presence of specific ligands. For instance, replacement of amide hydrogen by deuterium is extremely sensitive to environmental changes and the kinetics of exchange can be used to detect tertiary conformational changes in the protein structure. Moreover, in addition to the conformational parameters that can be deduced from the shape of the infrared spectra, the orientation of various parts of the molecule can be estimated with polarized IR. This allows more precise analysis of the general architecture of the membrane molecules within the biological membranes. The present review focuses on ATR-IR as an experimental approach of special interest for the study of the structure, orientation, and tertiary structure changes in peptides and membrane proteins.     

Redox-induced transitions in bovine cytochrome bc1 complex studied by perfusion-induced ATR-FTIR spectroscopy

Redox transitions in a film of detergent-purified bovine cytochrome bc(1) complex were investigated by perfusion-induced attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The technique provides a flexible method for generating redox-induced IR changes of components of bovine cytochrome bc(1) complex at a high signal:noise ratio. These IR redox difference spectra arise from perturbations of prosthetic groups and surrounding protein. Visible difference spectra were recorded synchronously using a light beam reflected from the exposed prism surface and provided a quantitative means of determining the redox transitions that were occurring. IR and visible redox difference spectra of iron-sulfur protein/cytochrome c(1), heme b(H), and heme b(L) were separated by selective reduction and/or oxidation that extends published data on the homologous bacterial enzyme. Several bands could be tentatively assigned to redox-sensitive modes of hemes and ubiquinone and changes in the surrounding protein by comparison with available data for bacterial bc(1) complex, other related heme proteins, and model compounds. Some tentative assignments of further signals to specific amino acids are made on the basis of known crystal structures.     

Interaction of a G protein-coupled receptor with a G protein-derived peptide induces structural changes in both peptide and receptor: a Fourier-transform infrared study using isotopically labeled peptides

G protein-coupled receptor signaling involves productive interaction between agonist-activated receptor and G protein. We have used Fourier-transform infrared difference spectroscopy to examine the interaction between the active Meta II state of the visual pigment rhodopsin with a peptide analogue corresponding to the C terminus of the alpha-subunit of the G protein transducin. Formation of the receptor-peptide complex evokes a spectral signature consisting of conformationally sensitive amide I and amide II difference bands. In order to distinguish between amide backbone contributions of the peptide and of the receptor moiety to the vibrational spectra, we employed complete (13)C,(15)N-labeling of the peptide. This isotopic labeling downshifts selectively the bands of the peptide, which can thus be extracted. Our results show that formation of the complex between the activated Meta II receptor state and the peptide is accompanied by structural changes of the peptide, and of the receptor, indicating that the conformation of the Meta II.peptide complex is different from that of Meta II. This result implies that the activated receptor state has conformational flexibility. Binding of the peptide to the activated receptor state stabilizes a substate that deviates from that stabilized only by the agonist.     

Ligand binding to the membrane-bound acetylcholine receptor from Torpedo marmorata: a complete mathematical analysis.

We have studied by means of equilibrium binding and kinetic experiments the interaction of the membrane-bound nicotinic acetylcholine receptor (nACHR) from Torpedo marmorata with [3H]acetylcholine and the fluorescent agonist NBD-5-acylcholine. In agreement with previous studies by others, we observed the preexistence, in the absence of ligand, of an equilibrium between two states of the nAChR, one with high affinity and the other with low affinity for agonist. As additional requirements for a minimal reaction scheme, we recognized (i) the existence of two ligand-binding sites, each of which may exist in two conformational states when occupied, and (ii) ligand-induced transitions between these conformations. Employing a special form of the allosteric model which considers these requirements, we then developed a suitable algorithm in order to simultaneously fit the whole set of equilibrium binding and kinetic data obtained for the two ligands. In this way we determined for a minimal model of the mechanism of action of the nAChR the complete set of rate constants and KD values involved. With these values available, we were able to simulate the rise and fall in the concentrations of individual receptor-ligand complexes and conformations occurring in the course of excitatory events at the electrocyte synapse. The membrane environment of the nAChR plays a decisive role with respect to the rates of conformational change of the nAChR occurring in the course of ligand interaction. Thus, artificial changes in membrane structure and composition can speed up by several orders of magnitude the rate of conformational change ("desensitization"). A proper structure of the surrounding membrane hence is a prerequisite for the physiological function of the membrane-embedded nAChR.     

Neuronal nitric oxide synthase ligand and protein vibrations at the substrate binding site. A study by FTIR

Improvements in sensitivity and data processing of Fourier transform infrared (FTIR) spectroscopy enable it to be used to detect changes in protein structure at the atomic level. This paper reports a study of neuronal nitric oxide synthase (nNOS) by FTIR difference spectroscopy in the 1000-2500 cm(-1) range where vibrational bands of ligands, prosthetic groups, and protein and amino acid side chains are found. We have exploited the photolyzable CO compound of the ferrous heme of nNOS to produce light-induced CO photolysis difference spectra and to compare spectra after hydrogen/deuterium exchange. In (reduced) minus (reduced plus CO) difference spectra, negative bands at 1931 and 1907 cm(-1) are observed due to photolysis of multiple forms of ferrous heme-ligated CO, similar to those observed by resonance Raman spectroscopy [Wang et al. (1997) Biochemistry 36, 4595-4606]. Photolysis of the ferrous heme CO compound is accompanied by hitherto unreported changes in the 1000-2000 cm(-1) region that arise from changes of protein backbone, substrate, amino acid side chain, and cofactor vibrations. Preliminary assignments of vibrations are made on the basis of frequencies and the effects of hydrogen/deuterium exchange, and in the light of known atomic structures.     

Internal dynamics of the nicotinic acetylcholine receptor in reconstituted membranes

The structure and 1H/2H exchange kinetics of affinity-purified nAChR reconstituted into egg phosphatidylcholine membranes with increasing levels of either dioleoylphosphatidic acid (DOPA) or cholesterol (Chol) have been examined using infrared spectroscopy. All spectra of the reconstituted nAChR membranes recorded after 72 h in 2H2O exhibit comparable amide I band shapes, suggesting a similar secondary structure for the nAChR in each lipid environment. Increasing levels of either DOPA or Chol, however, lead to an increasing intensity of the amide II band, indicating a decreasing proportion of nAChR peptide hydrogens that have exchanged for deuterium. Spectra recorded as a function of time after exposure of the nAChR to 2H2O show that the presence of either lipid slows down the 1H/2H exchange of those peptide hydrogens that normally exchange on the minutes to hours time scale. The slowing of peptide 1H/2H exchange correlates with both an increasing ability of the nAChR to undergo agonist-induced conformational change [Baenziger, J. E., Morris, M.-L., Darsaut, T. E., and Ryan, S. E. (1999) in preparation] and possibly a decreasing membrane fluidity. Our data suggest that lipid composition dependent changes in nAChR peptide 1H/2H exchange kinetics reflect altered internal dynamics of the nAChR. Lipids may influence protein function by changing the internal dynamics of integral membrane proteins.     

Changes in the secondary structure of apolipoprotein B-100 after Cu2+-catalysed oxidation of human low-density lipoproteins monitored by Fourier transform infrared spectroscopy

Fourier transform infrared (FTIR) spectra have been obtained of human low-density lipoproteins (LDL) in H2O and 2H2O buffers. The absorption bands are assigned to vibrations of the lipid and apolipoprotein B-100 components. The analysis of second-derivative spectra allowed an assignment of individual protein bands to alpha-helical, random, coil or beta-structure and beta-turn conformations. Changes in the FTIR spectra after Cu2+-catalysed oxidation of the LDL particles indicate that the structure of apolipoprotein B-100 becomes less ordered, with some alterations of alpha-helical and beta-turn conformation. The main beta-structure absorption at 1620 cm-1 is unaffected by oxidation. Taking into account the resistance to oxidation and the slow H-2H exchange it is suggested that the beta-structure is hidden from external factors whereas other structures are mostly present on the surface of the LDL particle. Oxidation affects mainly the surface region of apolipoprotein B-100 and leads to a structural rearrangement which consequently changes the receptor specificity of the LDL.     

Characterization of the conformational change in the M1 and M2 substates of bacteriorhodopsin by the combined use of visible and infrared spectroscopy.

A combination of visible and Fourier transform infrared (FTIR) spectroscopies is used to characterize the formation of the M1 and M2 substates of the bacteriorhodopsin photocycle in glucose-embedded, hydrated thin films. Difference FTIR bands in the amide I region verify the previously reported existence of a significant peptide backbone conformational change in the transition from M1 to M2. The visible absorption spectra demonstrate that contamination of the M-intermediate samples by L, N, or other non-M species should contribute negligibly to the observed changes in the amide I region, and this conclusion is supported by comparison of specific carboxyl group peaks with corresponding bands in published L and N FTIR difference spectra. Based upon spectroscopic results, an extension of the C-T Model (Fodor, S., Ames, J., Gebhard, R., van den Berg, E., Stoeckenius, W., Lugtenberg, J., and Mathies, R. (1988) Biochemistry 27, 7097-7101) is presented. The results of this work suggest that protein structural changes should be clearly visible in M-bR, difference Fourier density maps and that these structural changes may in turn elucidate how bacteriorhodopsin actively pumps ions across the purple membrane of Halobacterium halobium.     

Qualitative and quantitative analysis of the secondary structure of cytochrome C Langmuir-Blodgett films

A qualitative and quantitative analysis of the conformation of Langmuir-Blodgett (LB) dried films of cytochrome C on silicon wafers was performed by Fourier transform ir (FTIR) spectroscopy. A deconvolution procedure was applied to the amide I band analysis, in order to determine the percentage of the different secondary structures. Qualitative analysis was performed by examining difference spectra. Films obtained by spreading protein solutions at pH 7.4 and 1, dried at 25 and 100°C, on silicon wafers were also examined in order to detect spectral components associated with denatured protein domains, and to compare them with cytochrome C LB films. FTIR spectroscopy showed that the following important changes characterise LB film spectra: (a) the α-helix component is higher (its percentage is 57 and 54%) than the one estimated in dried film obtained by spreading the solutions at pH 7.4 on a silicon substrate (43%), (b) there is an increase in the intensity of bands attributed to protonated carboxy group bands, involved and not involved in the formation of hydrogen bonds, and a decrease in those attributed to deprotonated carboxy groups, (c) the intensity of several bands attributed to aromatic amino acids and aliphatic chains increases, and (d) bands due to O(SINGLEBOND)H stretching vibrations of crystallization water are present. These conformational changes could be induced by protein-protein interaction caused by the close packing of molecules that occurs during LB film formation; it cannot be excluded that they may be accompanied by partial changes in the tertiary structure of the protein. A preferential orientation of protein molecules in LB films is also a possibility. © 1997 John Wiley & Sons, Inc. Biopoly 42: 227–237, 1997     

Fourier transform infrared spectroscopy indicates a major conformational rearrangement in the activation of rhodopsin.

The study of the structural differences between rhodopsin and its active form (metarhodopsin II) has been carried out by means of deconvolution analysis of infrared spectra. Deconvolution techniques allow the direct identification of the spectral changes that have occurred, which results in a significantly different view of the conformational changes occurring after activation of the receptor as compared with previous difference spectroscopy analysis. Thus, a number of changes in the bands assigned to solvent-exposed domains of the receptor are detected, indicating significant decreases in extended (beta) sequences and in reverse turns, and increases in irregular/aperiodic sequences and in helices with a non-alpha geometry, whereas there is no decrease in alpha-helices. In addition to secondary structure conversions, qualitative alterations within a given secondary structure type are detected. These are seen to occur in both reverse turns and helices. The nature of this spectral change is of great importance, since a clear alteration in the helices bundle core is detected. All these changes indicate that the rhodopsin --> metarhodopsin II transition involves not a minor but a major conformational rearrangement, reconciling the infrared data with the energetics of the activation process.     

Light-induced changes in the chemical bond structure of light-harvesting complex II probed by FTIR spectroscopy

Light-harvesting complex II (LHC-II) regulates the light energy distribution between photosystem I and II in plants. This process is mediated by phosphorylation of the LHC-II protein, which depends on the oxidation state of photosynthetic electron carriers. In addition to this regulatory mechanism, it has recently been proposed that light can directly induce a conformational change in isolated LHC-II. To provide biophysical evidence for such a conformational change in the protein, we studied infrared absorbance changes in isolated LHC-II upon exposure to light flashes. Compared to the signals obtained with other proteins that exhibit well-characterized conformational changes, the signal in the LHC-II difference spectra is very weak. The position of the difference bands coincides with the main IR absorption bands of chlorophyll. We conclude that there are no detectable light-induced changes in the LHC protein structure and attribute the observed IR signals to light-induced chlorophyll degradation.     

FTIR difference spectroscopy of bacteriorhodopsin: Toward a molecular model

Bacteriorhodopsin (bR) is a light-driven proton pump whose function includes two key membrane-based processes, active transport and energy transduction. Despite extensive research on bR and other membrane proteins, these processes are not fully understood on the molecular level. In the past ten years, the introduction of Fourier transform infrared (FTIR) difference spectroscopy along with related techniques including time-resolved FTIR difference spectroscopy, polarized FTIR, and attenuated total reflection FTIR has provided a new approach for studying these processes. A key step has been the utilization of site-directed mutagenesis to assign bands in the FTIR difference spectrum to the vibrations of individual amino acid residues. On this basis, detailed information has been obtained about structural changes involving the retinylidene chromophore and protein during the bR photocycle. This includes a determination of the protonation state of the four membrane-embedded Asp residues, identification of specific structurally active amino acid residues, and the detection of protein secondary structural changes. This information is being used to develop an increasingly detailed picture of the bR proton pump mechanism.     

Attenuated total reflection Fourier transform infrared studies of redox changes in bovine cytochrome c oxidase: resolution of the redox Fourier transform infrared difference spectrum of heme a(3).

Attenuated total reflection Fourier transform infrared (ATR-FTIR) difference spectroscopy has been performed on samples of bovine cytochrome c oxidase that have been deposited as a thin film on the surface of a silicon microprism. The technique has several advantages over transmission methods in terms of amount of material required, the time required to reach sufficient optical stability, and the range of reactants that can be repetitively added and removed. The ATR-FTIR method has been used to record redox difference spectra of cytochrome c oxidase in the unligated and cyanide-ligated states. By subtraction of the spectra, the redox FTIR difference spectrum of heme a(3) can be resolved from those of the other metal centers. This difference spectrum is compared with available vibrational and Raman data on homologous oxidases and on heme A model compounds.     

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.