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Infrared reflection-absorption spectroscopy (IRRAS) intensities of the Amide I vibration are used to develop a quantitative approach for determining the Euler angles that describe the orientation of protein beta-sheets in aqueous monolayer films. A synthetic amphipathic peptide, Val-Glu-Val-Orn-Val-Glu-Val-Orn-Val-Glu-Val-Orn-Val-OH is used as a test case. The pattern of Amide I frequencies suggests that the molecule is organized as an antiparallel beta-sheet at the air/water interface. The model used to simulate the Amide I intensities reveals that the beta-sheet has a slight preferential alignment parallel to the direction of compression; i.e., deviation from uniaxial symmetry is observed. In addition, the sheet is found to lie flat on the aqueous surface, with (presumably) the polar side chains interacting with the aqueous subphase. Limitations and advantages of the theoretical approach are discussed.

IR reflection−absorption spectroscopy (IRRAS) is used to determine the orientation of carbonyl groups in behenic acid methyl ester (BME) and in sn2-13C 1,2 distearoylphosphatidylcholine (sn2-13C-DSPC) in aqueous monolayer films. The CO stretching contour of BME consists of three overlapped peaks arising from unhydrated, monohydrated, and doubly hydrated forms at 1737, 1721, and 1702 cm-1, respectively. Dichroic ratios constructed from the p- and s-polarized IRRAS intensities of the resolved components of the contour were simulated with the optical theory of V. L. Kuzmin.1,2 On the basis of valence bond and molecular modeling considerations, the carbonyl group is essentially perpendicular to the chain axes. The transition moment is determined by IRRAS to indeed lie along the CO bond for the unhydrated groups (thus providing good support for the theory) but changes direction for hydrated groups. A satisfactory fit between simulation and experiment could not be obtained by assuming a uniaxial distribution of...

The performance of a new accessory for measuring IR reflection-absorption (IRRAS) spectra at various angles of incidence from monolayer films of lipids is demonstrated. The device is comprised of an external variable angle reflectance accessory and a self-contained computer-controlled unit for varying the angle of incidence, both units being coupled to a custom-designed Langmuir trough. This combination provides for excellent experimental precision and reduction of interference from H2O vapor in the 1500–1800 cm−1 spectral region. A set of ∼20 s- and p-polarized IRRAS spectra over a range of angles of incidence from 25° to 70° may be acquired from a single monolayer film in ∼3 h. The performance of the device is demonstrated for two lipids as follows. First, the instrument precision is demonstrated through determination of the orientation of each of the chains in a fatty acid homogeneous ceramide 2 derivative. Interestingly, the orientation (tilt angles from the bilayer normal) of the individual chains differ, as determined from an isotopically-labeled derivative where the fatty acid chain is perdeuterated. The fatty acid chain is tilted at 24° from the normal to the bilayer, while the base chain is tilted at 18°. As a control, the average tilt angle for both ceramide 2 acyl chains has been determined from a fully proteated derivative to be 20°. The precision of the derived tilt angle varies from ∼1–3°. Second, the reduction of interference from water vapor in the 1500–1800 cm−1 is demonstrated for sn-2–13C=O-1,2 distearoylphosphatidylcholine, where spectra from the individual C=O bonds in the molecule are readily detected with good signal/noise and signal/water vapor ratios. The device provides a major advance in the determination of chain orientation by IRRAS and will facilitate the determination of functional group orientation.

Two-dimensional infrared (2D IR) spectroscopy has been shown to be a powerful tool for the analysis of spectra with highly overlapped bands, as often found in IR spectra of biological samples. To date, most 2D IR analyses have focused primarily on intensity changes of the bands under investigation. However, information concerning 2D IR characteristics of bands that change in position or width is sparse. We have thus simulated the effects of frequency and bandwidth changes on 2D IR spectra. In the synchronous plot of a band undergoing a frequency shift, two autopeaks and two crosspeaks are found at the initial and final positions, while the asynchronous plot exhibits two weaker crosspeaks for these positions and a stronger, somewhat elongated feature close to the diagonal. The latter feature is characteristic of a shifting band. Thus, to distinguish a frequency shift in a single band from intensity changes of two overlapped bands it is important to examine the asynchronous plot, since the synchronous plots exhibit comparable characteristics in both cases. A bandwidth change results in a series of crosspeaks. However, when bandwidth changes are coupled with either frequency shifts and/or intensity changes, the effect of the bandwidth change is reduced. Finally, it is shown that the resolution enhancement generally found for the asynchronous plot is accompanied by an error in the positions of the original spectral features as determined from 2D IR peaks. The magnitude of the error increases as the original spectral features approach each other in frequency. © 1996 John Wiley & Sons, Inc.

The current theory of pulmonary surfactant function requires that very low surface tension be achieved and maintained in the alveolar surface film during compression (expiration). To effect this condition, it has been hypothesized that the unsaturated and/or fluid components of surfactant are selectively excluded or "squeezed out" from mixed monolayers containing both saturated and unsaturated phospholipids, leaving a surface film of essentially pure 1,2-dipalmitoylphosphatidylcholine (DPPC). External reflection Fourier transform infrared (FT-IR) spectroscopy has been employed to quantitatively test this hypothesis. Mixed monolayer films of acyl chain-perdeuterated 1,2-dipalmitoylphosphatidylcholine (DPPC-d62) with 1,2-dioleoylphosphatidylglycerol (DOPG), 1-palmitoyl, 2-oleoylPG (POPG), 1,2-dipalmitoylPG (DPPG) were examined in situ at the air/water interface as a function of surface pressure. The relative intensities of CD2 (CH2) stretching vibrations of the deuterated (proteated) components permitted quantitative determination of the relative concentrations of each in the film. For 7:1 (mol:mol) mixtures of DPPC-d62/DOPG, progressive, selective squeeze out of up to about 90% of the PG component is observed over a range of surface pressures from about 51 to 68 mN/m. The extent of maximal PG squeeze out was reduced to 61% for a 7:1 (mol:mol) mixture of DPPC-d62/POPG. This phenomenon, which is at least partially reversible, appears to require relatively high rates of film compression. Squeeze out was reduced (< 20%) for 7:1 (mol:mol) mixtures of DPPC-d62/DPPG or for 7:3 mixtures of DPPC-d62/POPG. Squeeze out requires that the lipid mixture achieve surface pressures greater than about 50-60 mN/m along with unsaturation (or at least conformational disorder) in the acyl chains of the non-DPPC component.(ABSTRACT TRUNCATED AT 250 WORDS)

An FT-IR spectrophotometer has been interfaced to a miniaturized surface film apparatus for external reflection studies of insoluble monolayers in situ at the air/water interface. Signal-to-noise ratios of 200:1 were routinely achieved for the CH2 stretching vibrations of phospholipids. We have monitored, using the acyl chain symmetric CH2 stretching frequency near 2850 cm−1 as a structural probe, lipid conformational order changes that occur during the surface pressure-induced two-dimensional phase transition in monolayers of 1,2-dipalmitoylphosphatidylserine. In addition, the small volume of the miniaturized film apparatus (30 mL) permitted replacement of H2O with D2O in the subphase. This capability, in turn, permits the acquisition of spectral data in the amide I region of proteins. We report the first external reflection FT-IR spectrum of an insoluble protein monolayer. The protein studied is pulmonary surfactant SP-C.

Fourier transform infrared (FT-IR) spectroscopy and differential scanning calorimetry (DSC) have been used to elucidate the phase behavior of two binary lipid mixtures, acyl chain perdeuterated 1,2-dipalmitoylphosphatidylethanolamine (DPPE-d62)/1,2-dielaidoylphosphatidylcholine (DEPC) and acyl chain perdeuterated 1,2-dipalmitoylphosphatidylcholine (DPPC-d62)/1,2-dimyristoylphosphatidylethanolamine (DMPE). The former shows gel state immiscibility over most of the composition range. The FT-IR data indicate that one of the solid phases is essentially pure DEPC, while the other solid phase contains both lipids. The DPPC-d62/DMPE pair are miscible over the entire composition range. The use of deuterated lipids as one component in the mixture permits the melting characteristics of each component to be separately determined in the FT-IR experiment. The FT-IR data are used to assign the endotherms observed in the DSC to particular molecular components. For the DPPE-d62/DEPC system, two endotherms are observed at compositions between 10 and 67 mol% DPPE-d62. The lower transition is assigned to the DEPC component, while the higher event contains contributions to the enthalpy from both lipids in the mixture. The midpoint of the DEPC melting occurs substantially below that for DPPE-d62. For the miscible pair, each of the lipids melt over approximately the same temperature range. The complementary and consistent nature of the information available from FT-IR and from DSC is demonstrated from the current work.

Attenuated total reflectance Fourier transform infrared (ATR/FT-IR) spectroscopy is a powerful and widely used technique for obtaining molecular structural information from thin films of material. The ability of the method to provide orientational information from partially ordered samples such as lipid multibilayers (possibly with incorporated peptides) has resulted in several recent applications of the method to biophysical problems. In most ATR studies, the material under investigation is deposited as a dry film on an appropriate crystal, Ge or ZnSe being widely used for this purpose. Obviously, the biological relevance of dry films is questionable, as water often forms an integral part of the in vivo macromolecular assembly. It is evidently desirable to study biological samples under conditions of full hydration, where the effects of variation in pH and ionic strength and composition can be explored. Commercial ATR cells with water jackets are available, but these are expensive and fragile and have fairly large jacket volumes.

Although the determination of conformational disorder in biological membranes is an important goal of current membrane biophysics, a quantitative formulation has remained elusive. The current study describes a successful determination of trans-gauche isomerization at specific depths in phospholipid bilayers. The approach was suggested by Snyder and Poore (1) and utilizes the CD2 rocking modes of specifically deuterated hydrocarbons as conformational probes. This CD2 probe frequency is dependent on the conformation of the C-C-C skeleton of which the central carbon atom contains the CD2 moiety. The various conformations give rise to discrete (but weak) bands in the IR, as summarized below