Conformational changes in concanavalin A associated with demetallization and alpha-methylmannose binding studied by Fourier transform infrared spectroscopy

Infrared spectra of concanavalin A have been obtained both in the absence and in the presence of the metal ions, Mn2+ and Ca2+, and the saccharide, alpha-methylmannose. Second derivative calculations have been used to determine the frequencies of the different amide I and II components. In the demetallized protein dissolved in H2O buffer, absorptions in the amide I, II and III regions at 1695 and 1634, 1532 and 1237 cm-1, respectively, are assigned to beta-structure, while absorptions at 1563 and both 1318 and 1343 cm-1 are assigned to turns and bends. After deuterium exchange, the residual amide II maximum in the difference spectrum shifts from 1538 to 1563 cm-1, indicating that exchange is faster in the beta-structure than in the turns. In the presence of Mn2+ and Ca2+, the amide II band component at 1532 cm-1 shifts 4-6 cm-1 to higher wavenumbers, and the amide I band component at 1634 shifts 1 cm-1 in the same direction, both in H2O and 2H2O buffers, suggesting changes in the hydrogen-bonding network of a large portion of the protein, particularly in the beta-sheet regions. The addition of alpha-methylmannose increases the magnitude of exchange from 55% to above 90%. Comparison with existing X-ray crystallographic data has been made, and the usefulness of FT-IR to complement this technique is discussed.     

Polarized Fourier transform infrared spectroscopy of bacteriorhodopsin. Transmembrane alpha helices are resistant to hydrogen/deuterium exchange.

The secondary structure of bacteriorhodopsin has been investigated by polarized Fourier transform infrared spectroscopy combined with hydrogen/deuterium exchange, isotope labeling and resolution enhancement methods. Oriented films of purple membrane were measured at low temperature after exposure to H2O or D2O. Resolution enhancement techniques and isotopic labeling of the Schiff base were used to assign peaks in the amide I region of the spectrum. alpha-helical structure, which exhibits strong infrared dichroism, undergoes little H/D exchange, even after 48 h of D2O exposure. In contrast, non-alpha-helical structure, which exhibits little dichroism, undergoes rapid H/D exchange. A band at 1,640 cm-1, which has previously been assigned to beta-sheet structure, is found to be due in part to the C = N stretching vibration of protonated Schiff base of the retinylidene chromophore. We conclude that the membrane spanning regions of bR consist predominantly of alpha-helical structure whereas most beta-type structure is located in surface regions directly accessible to water.     

Infrared spectroscopic study of photoreceptor membrane and purple membrane. Protein secondary structure and hydrogen deuterium exchange

Infrared spectroscopy in the interval from 1800 to 1300 cm-1 has been used to investigate the secondary structure and the hydrogen/deuterium exchange behavior of bacteriorhodopsin and bovine rhodopsin in their respective native membranes. The amide I' and amide II' regions from spectra of membrane suspensions in D2O were decomposed into constituent bands by use of a curve-fitting procedure. The amide I' bands could be fit with a minimum of three theoretical components having peak positions at 1664, 1638, and 1625 cm-1 for bacteriorhodopsin and 1657, 1639, and 1625 cm-1 for rhodopsin. For both of these membrane proteins, the amide I' spectrum suggests that alpha-helix is the predominant form of peptide chain secondary structure, but that a substantial amount of beta-sheet conformation is present as well. The shape of the amide I' band was pH-sensitive for photoreceptor membranes, but not for purple membrane, indicating that membrane-bound rhodopsin undergoes a conformation change at acidic pH. Peptide hydrogen exchange of bacteriorhodopsin and rhodopsin was monitored by observing the change in the ratio of integrated absorbance (Aamide II'/Aamide I') during the interval from 1.5 to 25 h after membranes were introduced into buffered D2O. The fraction of peptide groups in a very slowly exchanging secondary structure was estimated to be 0.71 for bacteriorhodopsin at pD 7. The corresponding fraction in vertebrate rhodopsin was estimated to be less than or equal to 0.60. These findings are discussed in relationship to previous studies of hydrogen exchange behavior and to structural models for both proteins.     

Protein secondary structures in water from second-derivative amide I infrared spectra

Infrared spectra have been obtained for 12 globular proteins in aqueous solution at 20 degrees C. The proteins studied, which vary widely in the relative amounts of different secondary structures present, include myoglobin, hemoglobin, immunoglobulin G, concanavalin A, lysozyme, cytochrome c, alpha-chymotrypsin, trypsin, ribonuclease A, alcohol dehydrogenase, beta 2-microglobulin, and human class I major histocompatibility complex antigen A2. Criteria for evaluating how successfully the spectra due to liquid and gaseous water are subtracted from the observed spectrum in the amide I region were developed. Comparisons of second-derivative amide I spectra with available crystal structure data provide both qualitative and quantitative support for assignments of infrared bands to secondary structures. Band frequency assignments assigned to alpha-helix, beta-sheet, unordered, and turn structures are highly consistent among all proteins and agree closely with predictions from theory. alpha-Helix and unordered structures can each be assigned to only one band whereas multiple bands are associated with beta-sheets and turns. These findings demonstrate a method of analysis of second-derivative amide I spectra whereby the frequencies of bands due to different secondary structures can be obtained. Furthermore, the band intensities obtained provide a useful method for estimating the relative amounts of different structures.     

New insights into the self-assembly of insulin amyloid fibrils: an H-D exchange FT-IR study

The solvent protection of the amide backbone in bovine insulin fibrils was studied by FT-IR spectroscopy. In the mature fibrils, approximately 85 +/- 2% of amide protons are protected. Of those "trapped" protons, a further 25 +/- 2 or 35 +/- 2% is H-D exchanged after incubation for 1 h at 1 GPa and 25 degrees C or 0.1 MPa and 100 degrees C, respectively. In contrast to the native or unfolded protein, fibrils do not H-D exchange upon incubation at 65 degrees C. A complete deuteration of H(2)O-grown fibrils occurs when the beta-sheet structure is reassembled in a 75 wt % DMSO/D(2)O solution. Our findings suggest a densely packed environment around the amide protons involved in the intermolecular beta-sheet motive. In disagreement with the concept of "amyloid fibers as water-filled nanotubes" [Perutz, M. F., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 5591-5595], elution of D(2)O-grown fibrils with H(2)O is complete, which is reflected by the vanishing of D(2)O bending vibrations at 1214 cm(-)(1). This implies the absence of "trapped water" within insulin fibrils. The rigid conformations of the native and fibrillar insulin contrast with transient intermediate states docking at the fibrils' ends. Room-temperature seeding is accompanied by an accelerated H-D exchange in insulin molecules in the act of docking and integrating with the seeds, proving that the profound structural disruption is the sine qua non of forming an aggregation-competent conformation.     

External reflection FTIR of peptide monolayer films in situ at the air/water interface: experimental design, spectra-structure correlations, and effects of hydrogen-deuterium exchange.

A Fourier transform infrared spectrometer has been interfaced with a surface balance and a new external reflection infrared sampling accessory, which permits the acquisition of spectra from protein monolayers in situ at the air/water interface. The accessory, a sample shuttle that permits the collection of spectra in alternating fashion from sample and background troughs, reduces interference from water vapor rotation-vibration bands in the amide I and amide II regions of protein spectra (1520-1690 cm-1) by nearly an order of magnitude. Residual interference from water vapor absorbance ranges from 50 to 200 microabsorbance units. The performance of the device is demonstrated through spectra of synthetic peptides designed to adopt alpha-helical, antiparallel beta-sheet, mixed beta-sheet/beta-turn, and unordered conformations at the air/water interface. The extent of exchange on the surface can be monitored from the relative intensities of the amide II and amide I modes. Hydrogen-deuterium exchange may lower the amide I frequency by as much as 11-12 cm-1 for helical secondary structures. This shifts the vibrational mode into a region normally associated with unordered structures and leads to uncertainties in the application of algorithms commonly used for determination of secondary structure from amide I contours of proteins in D2O solution.     

A Raman spectroscopic study of hen egg yolk phosvitin: structures in solution and in the solid state

Laser Raman spectroscopy has been employed to study the structure of the hen egg yolk protein phosvitin in H2O and D2O solutions at neutral and acidic pH (pD) and in the solid state. The Raman data indicate an unusual conformation for phosvitin in neutral aqueous solution, which is deficient in both alpha-helix and conventional beta-sheet conformations. This unusual pH 7 structure is, however, largely converted to a beta-sheet conformation in strongly acidic media (pH less than 2). beta-Sheet is also the predominant secondary structure for phosvitin in the solid state, obtained by lyophilization of the protein from aqueous solution at neutral pH. The imidazolium rings of histidyl residues remain significantly protonated near neutrality, which suggests substantial elevation of the pK for imidazolium ring ionizations of phosvitin in aqueous solution. This may result from extensive ion-pair interactions involving positively charged histidines and negatively charged phosphoserines, which are prevalent in the phosvitin sequence. The present results suggest that antiparallel beta-sheets may not be the secondary structure most characteristic of native phosvitin (physiological pH), even though beta-sheet is the predominant conformation for phosvitin in acidic solutions (pH 1.5) and in the lyophilized solid. Phosvitin appears to be the first protein for which the major component to the Raman amide I band is centered near 1685 cm-1, which is 10-40 cm-1 higher than proteins heretofore examined in aqueous solution by Raman spectroscopy.     

Redox-induced conformational changes in myoglobin and hemoglobin: electrochemistry and ultraviolet-visible and Fourier transform infrared difference spectroscopy at surface-modified gold electrodes in an ultra-thin-layer spectroelectrochemical cell.

Using suitable surface-modified electrodes, we have developed an electrochemical system which allows a reversible heterogeneous electron transfer at high (approximately 5 mM) protein concentrations between the electrode and myoglobin or hemoglobin in an optically transparent thin-layer electrochemical (OTTLE) cell. With this cell, which is transparent from 190 to 10,000 nm, we have been able to obtain electrochemically-induced Fourier-transform infrared (FTIR) difference spectra of both proteins. Clean protein difference spectra between the redox states were obtained because of the absence of redox mediators in the protein solution. The reduced-minus-oxidized difference spectra are characteristic for each protein and arise from redox-sensitive heme modes as well as from polypeptide backbone and amino acid side chain conformational changes concomitant with the redox transition. The amplitudes of the difference bands, however, are small as compared to the total amide I absorbance, and correspond to approximately 1% (4%) of the reduced-minus-oxidized difference absorbance in the Soret region of myoglobin (hemoglobin) and to less than 0.1% of the total amide I absorbance. Some of the bands in the 1560-1490-cm-1 spectral regions could be assigned to side-chain vibrational modes of aromatic amino acids. In the conformationally sensitive spectral region between 1680 and 1630 cm-1, bands could be attributed to peptide C = O modes because of their small (2-5 cm-1) shift in 2H2O. A similar assignment could be achieved for amide II modes because of their strong shift in 2H2O.(ABSTRACT TRUNCATED AT 250 WORDS)     

Halogenated alcohols as solvents for proteins: FTIR spectroscopic studies.

Fourier transform infrared (FTIR) spectroscopy has been applied to investigate the secondary structure of proteins and polypeptides in halogenated alcohols. Each alcohol studied was able, as a pure liquid, to induce conversion of the beta-sheet protein concanavalin A into a predominantly alpha-helical configuration. In 2H2O/alcohol mixtures, helicogenisis was also apparent, decreasing in the order dichloroethanol greater than bromoethanol greater than trifluoroethanol greater than chloroethanol greater than fluoroethanol. At concentrations below those found to be helicogenic, disruption of the protein secondary structure by the alcohols resulted in pronounced aggregation. At concentrations insufficient to cause noticeable disruptions of the secondary structure at room temperature, the thermal stability of the protein was greatly reduced. We suggest the helicogenic effect exhibited by halogenated alcohols to be related to a combination of a relatively low dielectric constant and a high dipole moment, the latter causing disruption of the internal hydrogen bond networks and the former causing refolding to a helical configuration. The results presented here highlight the risk of using halogenated alcohols, both as solvents for proteins and as a test of the intrinsic capacity of proteins and peptides to adopt helical secondary structures.     

Beta-sheet and associated turn signatures in vibrational Raman optical activity spectra of proteins.

We have measured the aqueous solution vibrational Raman optical activity (ROA) spectra of concanavalin A, alpha-chymotrypsin, and beta-lactoglobulin, all of which are rich in beta-sheet, together with that of the model beta-turn peptide L-pro-L-leu-gly-NH2. Possible ROA signatures of antiparallel beta-sheet include a strong sharp positive band at approximately 1,313 cm-1 associated with backbone amide III C alpha H and NH deformations, and an amide I couplet, negative at low wavenumber and positive at high, centered at approximately 1,658 cm-1. Negative ROA bands in the range approximately 1,340-1,380 cm-1, which might originate in glycine CH2 deformations, appear to be characteristic of beta-turns. Our results provide further evidence that ROA is a more incisive probe of protein conformation than conventional vibrational spectroscopy, infrared, or Raman, because only those few vibrational coordinates within a given normal mode that sample the skeletal chirality directly contribute to the corresponding ROA band intensity.     

Dynorphin A (1-13) peptide NH groups are solvent exposed: FT-IR and 500 MHz 1H NMR spectroscopic evidence

FT-IR spectroscopic studies of dynorphin A(1-13) in H2O and D2O are utilized to derive the aqueous phase secondary structure of the opioid peptide. Resolution enhancement of the amide I region of dynorphin A(1-13) in H2O revealed a doublet at 1652 cm-1 and 1669 cm-1 which are interpreted as indicative of "unordered" and extended structures. From FT-IR and 1H NMR deuterium exchange studies, the peptide NH groups appeared to be solvent accessible which is suggestive of an essentially extended structure with aperiodically interwoven "unordered" structure. The results are consistent with Raman Spectroscopic (Rapaka et al., (1987) Int. J. Peptide Protein Res. 30:284-287) and 2D NMR studies (Huang et al. submitted), from our laboratory.     

Fourier transform infrared spectrometric analysis of protein conformation: effect of sampling method and stress factors.

Changes in the amide bands in Fourier transform infrared spectra of proteins are generally attributed to alterations in protein secondary structure. In this study spectra of five different globular proteins were compared in the solid and solution states recorded with several sampling techniques. Spectral differences for each protein were observed between the various sampling techniques and physical states, which could not all be explained by a change in protein secondary structure. For example, lyophilization in the absence of lyoprotectants caused spectral changes that could (partially) have been caused by the removal of hydrating water molecules rather than secondary structural changes. Moreover, attenuated total reflectance spectra of proteins in H2O were not directly comparable to transmission spectra due to the anomalous dispersion effect. Our study also revealed that the amide I, II, and III bands differ in their sensitivities to changes in protein conformation: For example, strong bands in the region 1620-1630 and 1685-1695 cm(-1) were seen in the amide I region of aggregated protein spectra. Surprisingly, absorbance of such magnitudes was not observed in the amide II and III region. It appears, therefore, that only the amide I can be used to distinguish between intra- and intermolecular beta-sheet formation. Considering the differing sensitivity of the different amide modes to structural changes, it is advisable to utilize not only the amide I band, but also the amide II and III bands, to determine changes in protein secondary structure. Finally, it is important to realize that changes in these bands may not always correspond to secondary structural changes of the proteins.     

Structure of the pore-forming transmembrane domain of a ligand-gated ion channel

The structure of the pore-forming transmembrane domain of the nicotinic acetylcholine receptor from Torpedo has been investigated by infrared spectroscopy. Treatment of affinity-purified receptor with either Pronase or proteinase K digests the extramembranous domains (roughly 75% of the protein mass), leaving hydrophobic membrane-imbedded peptides 3-6 kDa in size that are resistant to peptide (1)H/(2)H exchange. Infrared spectra of the transmembrane domain preparations exhibit relatively sharp and symmetric amide I and amide II band contours centered near 1655 and 1545 cm(-)1, respectively, in both (1)H(2)O and (2)H(2)O. The amide I band is very similar to the amide I bands observed in the spectra of alpha-helical proteins, such as myoglobin and bacteriorhodopsin, that lack beta structure and exhibit much less beta-sheet character than is observed in proteins with as little as 20% beta sheet. Curve-fitting estimates 75-80% alpha-helical character, with the remaining peptides likely adopting extended and/or turn structures at the bilayer surface. Infrared dichroism spectra are consistent with transmembrane alpha-helices oriented perpendicular to the bilayer surface. The evidence strongly suggests that the transmembrane domain of the nicotinic receptor, the most intensively studied ligand-gated ion channel, is composed of five bundles of four transmembrane alpha-helices.     

Comparative Fourier transform infrared spectroscopy study of cold-, pressure-, and heat-induced unfolding and aggregation of myoglobin

We studied the cold unfolding of myoglobin with Fourier transform infrared spectroscopy and compared it with pressure and heat unfolding. Because protein aggregation is a phenomenon with medical as well as biotechnological implications, we were interested in both the structural changes as well as the aggregation behavior of the respective unfolded states. The cold- and pressure-induced unfolding both yield a partially unfolded state characterized by a persistent amount of secondary structure, in which a stable core of G and H helices is preserved. In this respect the cold- and pressure-unfolded states show a resemblance with an early folding intermediate of myoglobin. In contrast, the heat unfolding results in the formation of the infrared bands typical of intermolecular antiparallel beta-sheet aggregation. This implies a transformation of alpha-helix into intermolecular beta-sheet. H/2H-exchange data suggest that the helices are first unfolded and then form intermolecular beta-sheets. The pressure and cold unfolded states do not give rise to the intermolecular aggregation bands that are typical for the infrared spectra of many heat-unfolded proteins. This suggests that the pathways of the cold and pressure unfolding are substantially different from that of the heat unfolding. After return to ambient conditions the cold- or pressure-treated proteins adopt a partially refolded conformation. This aggregates at a lower temperature (32 degrees C) than the native state (74 degrees C).     

Secondary structure in properdin of the complement cascade and related proteins: a study by Fourier transform infrared spectroscopy

Six structural repeat motifs of 58 amino acids are found in the sequence of both mouse and human properdins. Twelve more examples of the motif are available from the sequences of thrombospondin, the terminal complement components, and the thrombospondin-related anonymous protein. The averaged Robson and Chou-Fasman secondary structure predictions show that there are 57-66% turn and 19-38% beta-sheet structures in the typical repeat motif. The high amount of turn structure is consistent with Gly, Pro, Cys, and Ser being the four most abundant amino acid residues in properdin. Comparisons with sequences found in the circumsporozoite protein from several species of malaria parasites show that their sequences and secondary structures strongly coincide only in a 18-residue segment. Further secondary structure analysis utilized Fourier transform infrared spectroscopy of human properdin in 2H2O buffers. These show a broad amide I band that, after second-derivative and deconvolution calculations, is shown to be composed of several components. Two at 1633 and 1683 cm-1 are strong evidence for beta-sheet structure, although overlap from beta-turns can also contribute. The presence of beta-turn structure is indicated by absorptions at 1662-1675 and 1645 cm-1. The properdin structure contains substantial quantities of beta-sheet and beta-turn structures, which is consistent with the secondary structure predictions and amino acid compositions. The length of the repeat motif is estimated as 3.3-4.3 nm, and an estimated 14-22% of nonexchanged amide protons reside in properdin. This is suggestive of a high degree of solvent accessibility in the structure.     

Valinomycin and its interaction with ions in organic solvents, detergents, and lipids studied by Fourier transform IR spectroscopy.

The structure of valinomycin in a range of organic solvents of varying polarity and in detergent and lipid dispersions has been studied by Fourier transform ir spectroscopy. In solvents of low polarity such as chloroform, ir spectra of valinomycin are fully consistent with the bracelet structure proposed on the basis of nmr spectroscopy, showing a single narrow amide I component attributable to the presence of beta-turns and a single band arising from nonhydrogen-bonded ester C = O groups. K+ complexation results in a downward shift in the amide I band frequency, indicating an increase in the strength of the amide hydrogen bonds, along with a shift to lower frequencies of the ester C = O absorption due to a reduction in electron density in these bonds upon complexation. Identical results were obtained with NH4+, a finding not previously reported. In solvents of both medium (CHCl3/DMSO 3:1) and high (pure DMSO) polarity, we find evidence of significant disruption of the internal hydrogen-bonding network of the peptide and the appearance of a band suggesting the presence of free amide C = O groups. In such solvents, complexation with K+ and NH4+ was not observed. The structure of valinomycin in detergent micelles resembles that in nonpolar organic solvents. However, changes were found in the amide I and ester carbonyl maxima as 2H2O penetrated the micelle which suggest significant interaction between the solvent and peptide. Complexation with K+ was reduced in cationic detergent micelles as a result of a decrease in the effective K+ concentration due to charge repulsion at the micelle surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Infrared spectroscopy of a single cell--the human erythrocyte

Methods for obtaining the infrared spectrum of a single erythrocyte by infrared microscopy have been developed. The spectrum contains the amide I, II, and III bands characteristic of protein secondary structure near 1650, 1550, and 1300 cm-1, respectively. Bound carbon monoxide exhibits a readily measured band at 1951 cm-1 for 12C16O and 1907 cm-1 for 13C16O. Both amide and CO bands are similar to those found for purified hemoglobin A. Spectra can be obtained in H2O or D2O media under physiologically relevant conditions. Single cell infrared spectroscopy (SCIR) permits the qualitative and quantitative determination of differences among individual red cells. These results suggest many potential applications for SCIR for the measurements of properties of individual cells at the molecular level under physiologically relevant conditions.     

A study of the structure of human complement component factor H by Fourier transform infrared spectroscopy and secondary structure averaging methods

Fourier transform infrared spectroscopy was used to investigate the secondary structure of human complement component factor H in H2O and 2H2O buffers. The spectra show a broad amide I band which after second-derivative calculations is shown to be composed of three components at 1645, 1663, and 1685 cm-1 in H2O and at 1638, 1661, and 1680 cm-1 in 2H2O. The frequencies of these components are consistent with the existence of an extensive antiparallel beta-strand secondary structure. The exchange properties of the amide protons of factor H as measured in 2H2O buffers are rapid and lead to an estimate of NH proton nonexchange that is comparable with those for small globular proteins. Human factor H is constructed from a linear sequence of 20 short consensus repeats with a mean of 61 residues in each one. To investigate the secondary structure further, secondary structure predictions were carried out on the basis of an alignment scheme for 101 sequences for these repeats as found in human factor H and 12 other proteins. These predictions were averaged in order to improve the reliability of the calculations. Both the Robson and the Chou-Fasman methods indicate significant beta-structural contents. Residues 21-51 in the 61-residue repeat show a clear prediction of four strands of beta-structure and four beta-turns. A structural model based on antiparallel beta-strands in the secondary structure is proposed and discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of the secondary structure content of proteins in aqueous solutions from their amide I and amide II infrared bands. Comparison between classical and partial least-squares methods

A method for estimating protein secondary structure from infrared spectra has been developed. The infrared spectra of H2O solutions of 13 proteins of known crystal structure have been recorded and corrected for the spectral contribution of water in the amide I and II region by using the algorithm of Dousseau et al. [Dousseau, F., Therrien, M., & Pézolet, M. (1989) Appl. Spectrosc. 43, 538-542]. This calibration set of proteins has been analyzed by using either a classical least-squares (CLS) method or the partial least-squares (PLS) method. The pure-structure spectra calculated by the classical least-squares method are in good agreement with spectra of poly(L-lysine) in the alpha-helix, beta-sheet, and undefined conformations. The results show that the best agreement between the secondary structure determined by X-ray crystallography and that predicted by infrared spectroscopy is obtained when both the amide I and II bands are used to generate the calibration set, when the PLS method is used, and when it is assumed that the secondary structure of proteins is composed of only four types of structure: ordered and disordered alpha-helices, beta-sheet, and undefined conformation. Attempts to include turns in the secondary structure estimation have led to a loss of accuracy. The standard deviation of the difference between X-ray and infrared secondary structure estimates with this method is 4.8% for the alpha-helix, 3.7% for the beta-sheet, and 5.1% for the undefined structure, whereas the regression coefficients are 0.95, 0.96, and 0.56, respectively. The spectra of the calibration proteins were also recorded in 2H2O solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fourier transform infrared evidence for a predominantly alpha-helical structure of the membrane bound channel forming COOH-terminal peptide of colicin E1.

The structure of the membrane bound state of the 178-residue thermolytic COOH-terminal channel forming peptide of colicin E1 was studied by polarized Fourier transform infrared (FTIR) spectroscopy. This fragment was reconstituted into DMPC liposomes at varying peptide/lipid ratios ranging from 1/25-1/500. The amide I band frequency of the protein indicated a dominant alpha-helical secondary structure with limited beta- and random structures. The amide I and II frequencies are at 1,656 and 1,546 cm-1, close to the frequency of the amide I and II bands of rhodopsin, bacteriorhodopsin and other alpha-helical proteins. Polarized FTIR of oriented membranes revealed that the alpha-helices have an average orientation less than the magic angle, 54.6 degrees, relative to the membrane normal. Almost all of the peptide groups in the membrane-bound channel protein undergo rapid hydrogen/deuterium (H/D) exchange. These results are contrasted to the alpha-helical membrane proteins, bacteriorhodopsin, and rhodopsin.