Infrared reflection-absorption spectroscopy: principles and applications to lipid-protein interaction in Langmuir films

Abstract

Infrared reflection-absorption spectroscopy (IRRAS) of lipid/protein monolayer films in situ at the air/water interface provides unique molecular structure and orientation information from the film constituents. The technique is thus well suited for studies of lipid/protein interaction in a physiologically relevant environment. Initially, the nature of the IRRAS experiment is described and the molecular structure information that may be obtained is recapitulated. Subsequently, several types of applications, including the determination of lipid chain conformation and tilt as well as elucidation of protein secondary structure are reviewed. The current article attempts to provide the reader with an understanding of the current capabilities of IRRAS instrumentation and the type of results that have been achieved to date from IRRAS studies of lipids, proteins, and lipid/protein films of progressively increasing complexity. Finally, possible extensions of the technology are briefly considered.

Figure 1

IRRAS accessory currently in use at Rutgers University. The Wilhemy plate is placed on the right hand side of the trough. The reference channel is to the left of the barrier. The entire trough shuttles as indicated to sample the desired channel. The incident light is guided to the surface via three mirrors. The beam path is represented as a dashed line. The reflected light follows an equivalent optical path. The angles of incidence and reflection as well as the state of polarization are under computer control. (reprinted with permission (pending) from ).

Figure 2

IRRAS spectrum of the 4 component system DPPC/DPPG/cholesterol/SP-C on a D₂O subphase demonstrating the highS/N ratio achieved with current instrumentation in A) the methylene stretching region, 2800-3000 cm⁻¹ and B) the 1400-1800 cm⁻¹ region containing vibrations from the lipid and protein constituents as marked.

Figure 3

Precision and accuracy in quantitative IRRAS measurements. The molecule studied is a synthetic ceramide lipid model, whose structure is illustrated. A) Spectra of the CH₂ stretching region acquired with p-polarized radiation over a fairly narrow range of incident angles. Note the sign change of the RA at the Brewster angle. Further details in the text. B) Spectra of the CH₂ stretching region acquired with s-polarized radiation over a range of incident angles. C) Reproducibility of the p-polarized intensity of the CH₂ asymmetric stretch (2920 cm⁻¹) as a function of angle of incidence. Data for 4 independent films are overlaid. D) Reproducibility of the s-polarized intensity for the same band as a function of angle of incidence. (reprinted with permission (pending) from ).

Figure 4

Demonstration of the validity of the theoretical formalism used to calculate tilt angles of functional groups. The molecule is behenic acid methyl ester (structure shown). IRRAS spectral data (D₂O subphase) for p-polarized and s-polarized radiation are shown in A) and B), respectively. The complex C=O contour was resolved and the orientation of the 1737 cm⁻¹ band arising from unhydrated C=O groups was determined. The experimental p/s band intensity ratios are shown as a function of angle of incidence in C) along with the theoretical simulations for a 90° tilt angle. (reprinted with permission (pending) from ).

Figure 5

The first IRRAS determination of the determination of chain order for various phases of a lipid (DPPC) film. A) IRRAS spectra of DPPC monolayers at decreasing molecular areas as shown by the direction of the arrow B) The frequency of the asymmetric CH₂ stretching vibration plotted as a function of molecular area for the various DPPC phases as indicated. (reprinted with permission (pending) from and from Professor Rich Dluhy, University of Georgia).

Figure 6

Measured (■) and calculated (–) RA vs angle of incidence for the symmetric CD₂ stretching vibration of a monolayer of DPPC-d₆₂ on an H₂O subphase at 21°C and a surface pressure of 28mN/m. The best fit to the data was found using an acyl chain tilt angle of 26°. The calculated lines are shown along with the experimental data for s- and p-polarized light. (reprinted with permission (pending) from ). (Figure courtesy of Professor Arne Gericke, of Kent State University).

Figure 7

Sensitivity of the phosphate vibrations to hydration. A) Structure of Lipid A. B) S-polarized IRRAS spectra as a function of surface pressure, which increases from the top to the bottom spectrum. Spectra are offset for clarity. The phosphate vibrations at 1258, 1238 and 1225 cm⁻¹ represent unhydrated, monohydrated and dihydrated forms of particular phosphates in the molecule.

Figure 8

Langmuir trough design for examination of multilayer states of aqueous films: A) top, normal barrier design; bottom, embedded barrier design permitting acquisition of IRRAS spectra at high surface pressures. B) PM-IRRAS spectra of a DOPS monolayer at 26 mN/m (dashed line) and of a trilayer (solid line) at 44 mN/m in the alkyl chain methylene stretching region (top panel in B) and in the polar headgroup vibration range (bottom panel in B). (reprinted with permission (pending) from (Figure courtesy of Professor Bernard Desbat, CNRS, Talence Cedex, France).

Figure 9

Temporal stability of pulmonary surfactant SP-A NCRD Langmuir films in the absence (A) and presence (B) of a lipid A film at a surface pressure of 25 mN/m on a D₂O Ca²⁺-containing buffer.

Figure 10

Isotope labeling delineates the β-sheet region in the synthetic fragment NH₂-WLARALIKRIAQMIPKGA*LA*VA*VA*Q-VCR-COOH of pulmonary surfactant SP-B. Top spectrum, native SP-B; middle spectrum, unlabeled synthetic peptide sequence; bottom spectrum, peptide with alanines labeled (*following labeled residues). (reprinted with permission (pending) from ).

Figure 11

Determination of the orientation of Neuropeptide Y in a Langmuir film during compression. Characters adjacent to the p-polarized spectra in A), bottom panel correspond to pressures in the π-A isotherm shown in the inset (top left) as follows (a: π =0; b: 8mN/m; c:12 mN/m; d: 16 mN/m). Spectra were acquired for angles of incidence of 40° (solid line) and 60°, (dashed line). Also shown is a helical wheel representation of the peptide α-helix (black: hydrophobic residues; white hydrophilic residues; arrow: direction of the hydrophobic moment). B) Simulations of the Amide I and II bands (p-polarization for angles of incidence of 40° (top) and 60°(bottom) of neuropeptide Y for different tilt angles, φ (defined in the bottom panel), of the α-helix from the surface normal. (reprinted with permission (pending) from ) (Figure courtesy of Professor Mathias Lösche, Carnegie-Mellon University).

Figure 12

Pulmonary surfcatant SP-A-induced changes in the tilt angle of the DPPC acyl chains at two surface pressures and polarizations: A) π =25 mN/m, B) π =10 mN/m. DPPC films (o), DPPC/SP-A films (•), calculated curves A) pure DPPC (solid line) chain tilt angle= 32°; DPPC/SP-A (dashed line) chain tilt angle =31° B) pure DPPC (solid line) chain tilt angle= 35°; DPPC/SP-A (dashed line) chain tilt angle =28° (reprinted with permission (pending) from ).

Figure 13

A) PM-IRRAS spectra of a DMPC/gramicidin A layer (8:1 molar ratio) at the indicated film pressures on a D₂O subphase. B) Simulated PM-IRRAS spectra of the amide I band of gramicidin A at the indicated tilt angles on a D₂O subphase. (reprinted with permission (pending) from ) (Figure courtesy of Professor Horst Vogel, Ecole Polytechnique Federale de Lausanne and Dr. Peter Ulrich, SCIPROM, St-Sulpice, Switzerland).

Figure 14

Phospholipid interactions with Phospholipase D. A) PM-IRRAS spectra of different DPPC/DPPA mixtures at π = 40 mN/m. B) Integrated intensity of the phosphate vibrations as a function of the DPPC mole fractions in mixtures with DPPA. (•) sum of the symmetric PO₂⁻ and CO(P) modes, (▲) antisymmetric PO₂⁻ vibration band. C) hydrolysis yield catalyzed by 25 (•) and 255 (■) units of Phospholipase D. (reprinted with permission (pending) from and Professor Gerald Brezesinski, Max Planck Institute of Colloids and Interfaces, Golm/Potsdam, Germany).

Figure 15

IRRAS spectra (1765-1540 cm⁻¹) of human SP-D NCRD adsorption to Rd₁ LPS monolayers initially compressed to (A) π = 10 mN/m and (B) π = 25 mN/m. In each panel, the spectrum labeled: (1) was acquired prior to protein injection, (2) following protein injection under the lipid monolayer and pressure equilibration, (3) 10 min following subphase injection of an EDTA solution, and (4) 1 h after the EDTA injection. Surface pressures at the various stages are noted. The lipid C=O at ~1735 cm⁻¹ remains essentially unchanged under the various conditions demonstrating the stability of the LPS monolayer. A weak, broad Amide I feature (~1620-1650 cm⁻¹) arises from LPS in (1), both panels, but is overwhelmed by the intense Amide I band resulting from adsorbed SP-D in (2) and (3). Details of the EDTA injection are discussed in the text. (reprinted with permission (pending) from ).

Figure 16

Comparison of dichroic ratios of Amide I intensities at 1654 cm⁻¹ as a function of angle of incidence from experimental (symbols) versus simulated (lines) data to determine the orientation of the neck region in SP-D NCRD under various conditions. Experimental values were obtained from SP-D adsorption to: (○) a clean air/water interface, (● ) an LPS monolayer at Π = 10 mN/m, and (△) an LPS monolayer at Π = 25 mN/m. The mean and standard deviation for four experiments are shown for the π= 10 mN/m experiment, and averages for two experiments are shown for the two remaining datasets. Dichroic ratios are plotted from simulations conducted at particular neck axis tilt angles (0, 20 45, 90°) as marked. (reprinted with permission (pending) from ).

Figure 17

Poly-L Lactic acid spectrum obtained with PA-IRRS on a water subphase at 45° angle of incidence. Total acquisition time : 10.8 s for 1000 frames; total integration time :1.5 s. (reprinted with permission (pending) from and from Professor John Rabolt, University of Delaware).