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.     

Effect of temperature, pH, and metal ion binding on the secondary structure of bacteriorhodopsin: FT-IR study of the melting and premelting transition temperatures

We have measured the temperature dependence of the FT-IR spectra of bacteriorhodopsin (bR) as a function of the pH and of the divalent cation regeneration with Ca(2+) and Mg(2+). It has been found that although the irreversible melting transition shows a strong dependence on the pH of the native bR, the premelting reversible transition at 78-80 degrees C shows very little variation over the pH range studied. It is further shown that the acid blue bR shows a red-shifted amide I spectrum at physiological temperature, which shows a more typical alpha-helical frequency component at 1652 cm(-)(1) and could be the reason for the observed reduction of its melting temperature and lack of an observed premelting transition. Furthermore, the thermal transitions for Ca(2+)- and Mg(2+)-regenerated bR (Ca-bR and Mg-bR, respectively) each show a premelting transition at the same 78-80 degrees C temperature as the native purple membrane, but the irreversible melting transition has a slight dependence on the cation identity. The pH dependence of the Ca(2+)-regenerated bR is studied, and neither transition varies over the pH range studied. These results are discussed in terms of the cation contribution to the secondary structural stability in bR.     

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.     

FTIR analysis of the SII540 intermediate of sensory rhodopsin II: Asp73 is the Schiff base proton acceptor

Sensory rhodopsin II (SRII), a repellent phototaxis receptor found in Halobacterium salinarum, has several homologous residues which have been found to be important for the proper functioning of bacteriorhodopsin (BR), a light-driven proton pump. These include Asp73, which in the case of bacteriorhodopsin (Asp85) functions as the Schiff base counterion and proton acceptor. We analyzed the photocycles of both wild-type SRII and the mutant D73E, both reconstituted in Halobacterium salinarum lipids, using FTIR difference spectroscopy under conditions that favor accumulation of the O-like, photocycle intermediate, SII540. At both room temperature and -20 degrees C, the difference spectrum of SRII is similar to the BR-->O640 difference spectrum of BR, especially in the configurationally sensitive retinal fingerprint region. This indicates that SII540 has an all-trans chromophore similar to the O640 intermediate in BR. A positive band at 1761 cm-1 downshifts 40 cm-1 in the mutant D73E, confirming that Asp73 undergoes a protonation reaction and functions in analogy to Asp85 in BR as a Schiff base proton acceptor. Several other bands in the C=O stretching regions are identified which reflect protonation or hydrogen bonding changes of additional Asp and/or Glu residues. Intense bands in the amide I region indicate that a protein conformational change occurs in the late SRII photocycle which may be similar to the conformational changes that occur in the late BR photocycle. However, unlike BR, this conformational change does not reverse during formation of the O-like intermediate, and the peptide groups giving rise to these bands are partially accessible for hydrogen/deuterium exchange. Implications of these findings for the mechanism of SRII signal transduction are discussed.     

The role of the native lipids and lattice structure in bacteriorhodopsin protein conformation and stability as studied by temperature-dependent Fourier transform-infrared spectroscopy

We report the effect of partial delipidation and monomerization on the protein conformational changes of bacteriorhodopsin (bR) as a function of temperature. Removal of up to 75% of the lipids is known to have the lattice structure of the purple membrane, albeit as a smaller unit cell, whereas treatment by Triton monomerizes bR into micelles. The effects of these modifications on the protein secondary structure is analyzed by monitoring the protein amide I and amide II bands in the Fourier transform-infrared (FT-IR) spectra. It is found that removal of the first 75% of the lipids has only a slight effect on the secondary structure at physiological temperature, whereas monomerizing bR into micelles alters the secondary structure considerably. Upon heating, the bR monomer is found to have a very low thermal stability compared with the native bR with its melting point reduced from 97 to 65 degrees C, and the pre-melting transition in which the protein changes conformation in native bR at 80 degrees C could not be observed. Also, the N[bond]H to N[bond]D exchange of the amide II band is effectively complete at room temperature, suggesting that there are no hydrophobic regions that are protected from the aqueous medium, possibly explaining the low thermal stability of the monomer. On the other hand, 75% delipidated bR has its melting temperature close to that of the native bR and does have a pre-melting transition, although the pre-melting transition occurs at significantly higher temperature than that of the native bR (91 degrees C compared with 80 degrees C) and is still reversible. Furthermore, we have also observed that the reversibility of this pre-melting transition of both native and partially delipidated bR is time-dependent and becomes irreversible upon holding at 91 degrees C between 10 and 30 min. These results are discussed in terms of the lipid and lattice contribution to the protein thermal stability of native bR.     

Nanosecond retinal structure changes in K-590 during the room-temperature bacteriorhodopsin photocycle: picosecond time-resolved coherent anti-stokes Raman spectroscopy.

Time-resolved vibrational spectra are used to elucidate the structural changes in the retinal chromophore within the K-590 intermediate that precedes the formation of the L-550 intermediate in the room-temperature (RT) bacteriorhodopsin (BR) photocycle. Measured by picosecond time-resolved coherent anti-Stokes Raman scattering (PTR/CARS), these vibrational data are recorded within the 750 cm-1 to 1720 cm-1 spectral region and with time delays of 50-260 ns after the RT/BR photocycle is optically initiated by pulsed (< 3 ps, 1.75 nJ) excitation. Although K-590 remains structurally unchanged throughout the 50-ps to 1-ns time interval, distinct structural changes do appear over the 1-ns to 260-ns period. Specifically, comparisons of the 50-ps PTR/CARS spectra with those recorded with time delays of 1 ns to 260 ns reveal 1) three types of changes in the hydrogen-out-of-plane (HOOP) region: the appearance of a strong, new feature at 984 cm-1; intensity decreases for the bands at 957 cm-1, 952 cm-1, and 939 cm-1; and small changes intensity and/or frequency of bands at 855 cm-1 and 805 cm-1; and 2) two types of changes in the C-C stretching region: the intensity increase in the band at 1196 cm-1 and small intensity changes and/or frequency shifts for bands at 1300 cm-1 and 1362 cm-1. No changes are observed in the C = C stretching region, and no bands assignable to the Schiff base stretching mode (C = NH+) mode are found in any of the PTR/CARS spectra assignable to K-590. These PTR/CARS data are used, together with vibrational mode assignments derived from previous work, to characterize the retinal structural changes in K-590 as it evolves from its 3.5-ps formation (ps/K-590) through the nanosecond time regime (ns/K-590) that precedes the formation of L-550. The PTR/CARS data suggest that changes in the torsional modes near the C14-C15 = N bonds are directly associated with the appearance of ns/K-590, and perhaps with the KL intermediate proposed in earlier studies. These vibrational data can be primarily interpreted in terms of the degree of twisting of the C14-C15 retinal bond. Such twisting may be accompanied by changes in the adjacent protein. Other smaller, but nonetheless clear, spectral changes indicate that alterations along the retinal polyene chain also occur. The changes in the retinal structure are preliminary to the deprotonation of the Schiff base nitrogen during the formation of M-412. The time constant for the ps/ns K-590 transformation is estimated from the amplitude change of four vibrational bands in the HOOP region to be 40-70 ns.     

Infrared spectra of erythrocyte shadows in the region of the amide I and amide II bands following microwave irradiation]

No beta-structures of protein molecules were observed by IR-spectra of intact erythrocyte shadows. Ultra high frequency irradiation in the range of 1009 mHz intensity of 45 mW/cm3 results in small conformational reconstructions of molecules in the membrane, but it does not induce a notable transition of alpha-helix or coil into beta-structure. A decrease of the intensity of lipid band by 1740 cm-1 is shown up at the spectra. Deuterium exchange for 36--38 min shows that the transition of the band amide II near 1540 cm-1 into the band 1450 cm-1 proceeds faster under UHF irradiation than in the control. The effects observed are in a direct relation-ship with the intensity of UHF-field and disappear at the intensities of 5--8 mW/cm3 and lower.     

Investigation of secondary and tertiary structural changes of cytochrome c in complexes with anionic lipids using amide hydrogen exchange measurements: an FTIR study.

The structure of cytochrome c bound to anionic lipid membranes composed of dimyristoyl, dipalmitoyl, or dioleoyl phosphatidylglycerols, or of bovine heart cardiolipin, has been investigated by Fourier transform infrared spectroscopy. Only small changes in secondary structure, as registered by the amide I band of cytochrome c, were observed upon binding at temperatures below that of denaturation of the protein, and these were not coupled to the thermotropic phase transitions of the lipid. The denaturation temperature of the protein decreased by approximately 25-30 degrees upon binding, in a progression which correlated with that of the lipid phase transition temperatures, being approximately 7 degrees lower for complexes with dioleoyl than with dipalmitoyl phosphatidylglycerol. Large changes in the amide proton exchange characteristics, as monitored by the spectral shifts in the amide I band of the protein in D2O, were observed on binding cytochrome c to the lipid membranes. For the slowly exchanging population, the amide deuteration rates of the free protein were nearly independent of temperature, whereas those of the bound protein increased by up to two orders of magnitude over the temperature range from 10 to 40 degrees C. In addition, the extent of exchange differed between the bound and unbound protein. A structural transition in the bound protein was detected as a discontinuous step in Arrhenius plots of the deuterium exchange rates which occurred at a temperature in the region of 22 to 29 degrees C, depending on the lipid, far below that of denaturation. The temperature of this transition was determined by the physical state of the lipid, being 7 degrees lower for the lipids in the fluid state than for those in the gel state, and, for complexes with dimyristoyl phosphatidylglycerol, occurred at an intermediate temperature, being controlled by the lipid chain-melting transition at 27-28 degrees C. These results provide evidence for a coupling of the tertiary structure of the membrane-bound protein with the physical state of the membrane lipids.     

Fourier transform infrared spectroscopy and site-directed isotope labeling as a probe of local secondary structure in the transmembrane domain of phospholamban.

Phospholamban is a 52-amino acid residue membrane protein that regulates Ca(2+)-ATPase activity in the sarcoplasmic reticulum of cardiac muscle cells. The hydrophobic C-terminal 28 amino acid fragment of phospholamban (hPLB) anchors the protein in the membrane and may form part of a Ca(2+)-selective ion channel. We have used polarized attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy along with site-directed isotope labeling to probe the local structure of hPLB. The frequency and dichroism of the amide I and II bands appearing at 1658 cm-1 and 1544 cm-1, respectively, show that dehydrated and hydrated hPLB reconstituted into dimyristoylphosphatidycholine bilayer membranes is predominantly alpha-helical and has a net transmembrane orientation. Specific local secondary structure of hPLB was probed by incorporating 13C at two positions in the protein backbone. A small band seen near 1614 cm-1 is assigned to the amide I mode of the 13C-labeled amide carbonyl group(s). The frequency and dichroism of this band indicate that residues 39 and 46 are alpha-helical, with an axial orientation that is approximately 30 degrees relative to the membrane normal. Upon exposure to 2H2O (D2O), 30% of the peptide amide groups in hPLB undergo a slow deuterium/hydrogen exchange. The remainder of the protein, including the peptide groups of Leu-39 and Leu-42, appear inaccessible to exchange, indicating that most of the hPLB fragment is embedded in the lipid bilayer. By extending spectroscopic characterization of PLB to include hydrated, deuterated as well as site-directed isotope-labeled hPLB films, our results strongly support models of PLB that predict the existence of an alpha-helical hydrophobic region spanning the membrane domain.     

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.     

Picosecond dynamics of bacteriorhodopsin, probed by time-resolved infrared spectroscopy.

The photoinduced reaction cycle of bacteriorhodopsin (BR) has been studied by means of a recently developed picosecond infrared spectroscopic method at ambient temperature. BR - K difference spectra between 1560 and 1700 cm-1 have been recorded at delay times from 100 ps to 14 ns. The spectrum remains unchanged during this period. The negative difference OD band at 1660 cm-1 indicates the peptide backbone responds within 50 ps. A survey in the region of carboxylic side chain absorption around 1740 cm-1 reveals that perturbations of those groups, present in low-temperature FTIR spectra, are not observable within 10 ns, suggesting a slow conformational change.     

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.     

Identification of structural markers for vitamin B12 and other corrinoid derivatives in solution using FTIR spectroscopy

The identification of structural markers for B12/protein interactions is crucial to a complete understanding of vitamin B12 transport and metabolic reaction mechanisms of B12 coenzymes. Fourier transform infrared spectroscopy can provide direct measurements of changes in the side chains and corrin ring resulting from B12/protein interactions. Using FTIR spectroscopy in various solvent systems, we have identified structural markers for corrinoids in the physiological state. We assign the major band (denoted B), which occurs at ca. 1630 cm-1 in D2O and ca. 1675 cm-1 in ethanol, to the amide I C=O stretching mode of the propionamide side chains of the corrin ring. The lower frequency of band B in D2O versus ethanol is due to the greater hydrogen-bonding properties of D2O that stabilize the charged amide resonance form. Since the propionamides are known to be important in protein binding, band B is a suitable marker for monitoring the interaction of these side chains with proteins. We assign bands at ca. 1575 and 1545 cm-1 (denoted C and D) as breathing modes of the corrin ring on the basis of the bands' solvent independence and their sensitivity to changes in axial ligation. As the sigma-donating strength of the axial ligands increases, the frequencies of bands C and D decrease, possibly indicating a lengthening of the corrin conjugated system. Band A, the known cyanide stretching frequency at ca. 2130 cm-1, probes the cobalt-carbon distance in cyanocorrinoids. As the frequency of band A increases, the cobalt-carbon bond strength should decrease.     

Water structural changes in the L and M photocycle intermediates of bacteriorhodopsin as revealed by time-resolved step-scan Fourier transform infrared (FTIR) spectroscopy

In previous Fourier transform infrared (FTIR) studies of the photocycle intermediates of bacteriorhodopsin at cryogenic temperatures, water molecules were observed in the L intermediate, in the region surrounded by protein residues between the Schiff base and Asp96. In the M intermediate, the water molecules had moved away toward the Phe219-Thr46 region. To evaluate the relevance of this scheme at room temperature, time-resolved FTIR difference spectra of bacteriorhodopsin, including the water O-H stretching vibration frequency regions, were recorded in the micro- and millisecond time ranges. Vibrational changes of weakly hydrogen-bonded water molecules were observed in L, M, and N. In each of these intermediates, the depletion of a water O-H stretching vibration at 3645 cm-1, originating from the initial unphotolyzed bacteriorhodopsin, was observed as a trough in the difference spectrum. This vibration is due to the dangling O-H group of a water molecule, which interacts with Asp85, and its absence in each of these intermediates indicates that there is perturbation of this O-H group. The formation of M is accompanied by the appearance of water O-H stretching vibrations at 3670 and 3657 cm-1, the latter of which persists to N. The 3670 cm-1 band of M is due to water molecules present in the region surrounded by Thr46, Asp96, and Phe219. The formation of L at 298 K is accompanied by the perturbations of Asp96 and the Schiff base, although in different ways from what is observed at 170 K. Changes in a broad water vibrational feature, centered around 3610 cm-1, are kinetically correlated with the L-M transition. These results imply that, even at room temperature, water molecules interact with Asp96 and the Schiff base in L, although with a less rigid structure than at cryogenic temperatures.     

Oriented secondary structure in integral membrane proteins. I. Circular dichroism and infrared spectroscopy of cytochrome oxidase in multilamellar films.

The circular dichroism (CD) of cytochrome oxidase in solution indicates the presence of both alpha-helix (approximately 37%) and B-sheet (approximately 18%). In oriented films generated by the isopotential spin-dry method, the CD measured normal to the film shows a marked decrease in the negative bands at 222 and 208 nm, and a decrease and red shift in the positive band near 195 nm, relative to solution spectra. These features are characteristic of alpha-helices oriented with their helix axes along the direction of light propagation. A quantitative estimate of the orientation, based on the ratio of the rotational strengths of the 208-nm band in the film and in solution, leads to an average angle between the helix axis and the normal to the film, phi alpha of approximately 39 degrees. A method for analyzing infrared (IR) linear dichroism is developed that can be applied to proteins with comparable amounts of alpha-helix and beta-sheet. From analysis of the amide I band, phi alpha is found to lie between 20 and 36 degrees, depending on the angle that the amide I transition moment forms with the helix axis. A survey of the literature on the amide I transition moment direction indicates that a value of approximately 27 degrees is appropriate for standard alpha-helical systems, such as those in cytochrome oxidase. A larger value, near 40 degrees, is reasonable for systems that have distorted alpha-helices, as evidenced by amide I frequencies above 1,660 cm-1, as is the case of bacteriorhodopsin. This conclusion supports phi alpha approximately 36 degrees from IR linear dichroism, in agreement with the CD results. Linear dichroism in the amide I and amide II region indicates that the beta-sheet in cytochrome oxidase is oriented with the carbonyl groups nearly parallel to the plane of the membrane and the chain direction inclined at approximately 40 degrees to the normal. Comparison of these results with tentative identification of transmembrane helices from sequence data suggests that either some of the transmembrane helices are inclined at an unexpectedly large angle to the normal, or the number of such helices has been overestimated. Some putative transmembrane helices may be beta-strands spanning the membrane.     

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.     

Peptide-chain secondary structure of bacteriorhodopsin.

Ultraviolet circular dichroism spectroscopy in the interval from 190 to 240 nm and infrared spectroscopy in the region of the amide I band (1,600 cm-1 to 1,700 cm-1) has been used to estimate the alpha-helix content and the beta-sheet content of bacteriorhodopsin. Circular dichroism spectroscopy strongly suggests that the alpha-helix content is sufficient for only five helices, if each helix is composed of 20 or more residues. It also suggests that there is substantial beta-sheet conformation in bacteriorhodopsin. The presence of beta-sheet secondary structure is further suggested by the presence of a 1,639 cm-1 shoulder on the amide I band in the infrared spectrum. Although a structural model consisting of seven alpha-helical rods has been generally accepted up to this point, the spectroscopic data are more consistent with a model consisting of five alpha-helices and four strands of beta-sheet. We note that the primary amino acid sequence can be assigned to segments of alpha-helix and beta-sheet in a way that does not require burying more than two charged groups in the hydrophobic membrane interior, contrary to the situation for any seven-helix model.     

Topography of the membrane-binding domain of cytochrome b5 in lipids by Fourier-transform infrared spectroscopy

Fourier-transform infrared spectroscopy was used to examine the secondary structure of the membrane-binding domain (nonpolar peptide) of rabbit liver cytochrome b5 in D2O and in the presence of phospholipids and deoxycholate. In all situations, the predominant structure was alpha helix, but an examination of the components of the amide I band in the spectrum of the nonpolar peptide showed that the major peak was shifted from 1655 cm-1 in the lipids to 1650 cm-1 in deoxycholate. This shift to lower frequency, together with a decrease in intensity of the amide II band, is indicative of N-H to N-D exchange of the peptide backbone. A semiquantitative analysis indicated that the alpha helix of the peptide is over 95% exchanged in the presence of deoxycholate but is only 10% exchanged in the presence of lipid. These data suggest that the membrane-inserted portion of the peptide is alpha helical and is largely protected from N-H to N-D exchange by the bilayer. We suggest that this technique appears to provide a general method for determining the type of secondary structure involved in membrane interaction and the percentage of this structure which is involved in the interaction.     

Characterization by FTIR spectroscopy of the photoreduction of the primary quinone acceptor QA in photosystem II.

Molecular changes associated with the photoreduction of the primary quinone acceptor Qa of photosystem II have been characterized by Fourier transform infrared spectroscopy. This reaction was light-induced at room temperature on photosystem II membranes in the presence of hydroxylamine and diuron. A positive signal at 1478 cm-1 is assigned to the C---O stretching mode of the semiquinone anion, and can be correlated to the negative C=O mode(s) of the neutral QA at 1645 cm-1 and/or 16 cm-1. Analogies with bacterial reaction center are found in the amide I absorption range at 1672 cm-1, 1653 cm-1 and 1630 cm-1. The stabilization of QA- does not result from a large protein conformation change, but involves perturbations of several amino acid vibrations. At 1658 cm-1, a negative feature sensitive to 1H-2H exchange is tentatively assigned to a NH2 histidine mode, while tryptophan D2252 could contribute to the signal at 1560/1550 cm-1.     

Polarized infrared spectroscopy of oriented purple membrane.

Polarized Fourier transform infrared spectroscopy has been used to study the structure of purple membrane from Halobacterium halobium. Membranes were oriented by drying a suspension of membrane fragments onto Irtran-4 slides. Dichroism measurements of the amide I, II and A peaks were used to find the average spatial orientation of the bacteriorhodopsin alpha-helices. By deriving a function that relates the observed dichroism to the orientational order parameters for the peptide groups, helical axis distribution, and mosaic spread of the membranes, the average orientation of the alpha-helices was found to lie in a range of less than 26 degrees away from the membrane normal, agreeing with electron microscopic measurements. The frequency of the amide I and A peaks is at least 10 cm-1 higher than values found for most alpha-helical polypeptides and proteins. This may indicate that bacteriorhodopsin contains distorted alpha-helical conformations.