Joseph W. Brauner

External Infrared Reflection Absorption Spectrometry of Monolayer Films at the Air-Water Interface

The theory and practice of external infrared reflection absorption spectrometry (IRRAS) as applied to monomolecular films at the air-water interface are reviewed. The observed IR frequencies for films of amphiphilic species provide information about the conformational states of the hydrocarbon chains and the hydrogen bonding and ionization states of the polar head groups, under conditions of controlled surface pressure. Determination of molecular orientation is also feasible and requires detailed consideration of the reflection-absorption properties of the three- phase (air-monolayer-water) system. Current theoretical approaches are described. Applications of IRRAS to the study of single- and double-chain amphiphiles and proteins are reviewed, and initial excursions into biochemistry (interfacial enzyme catalysis) and physiology (pulmonary surfactant function) are reported.

A Quantitative Reconstruction of the Amide I Contour in the IR Spectra of Globular Proteins: From Structure to Spectrum

The Amide I contours of six globular proteins of varied secondary structure content along with a peptide model for collagen and pulmonary surfactant protein C have been simulated very closely by using a modified GF matrix method. The starting point for the method uses the three-dimensional structure as obtained from the Protein Data Bank. Elements of the interactions between peptide groups (e.g., transition dipole coupling) are very sensitive to tertiary structure, thus the current formalism demonstrates that the Amide I contour may be useful for a more detailed probe of 3-D conformation that goes beyond the traditional use of this band to probe the percentages of particular elements of secondary structure. For example, postulated changes to a known structure can be tested by comparing the new simulated band to the experimental band. A number of refinements to the transition dipole interaction calculation have been made. Most of the important interactions between the C=O oscillators that define the Amide I mode appear to have been identified, including through space transition dipole coupling, through valence bond and through hydrogen bond coupling. The eigenvector matrix produced by the method permits the contribution of each peptide group to the spectrum to be precisely determined. Analysis of the results shows that the often-used structure-frequency correlations are at best approximate and at worst misleading. The subbands from helices, sheets, turns, and loops are much broader and more overlapped than has been commonly assumed. Furthermore, the traditional alpha-helical marker band may be substantially distorted in short segments. Difference spectra based on isotope editing, a technique thought capable of revealing the spectral contributions of individual peptide groups, are shown to be prone to misinterpretation.

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.

Calcium ion interactions with insoluble phospholipid monolayer films at the A/W interface. External reflection-absorption IR studies

External reflection Fourier transform infrared (FT-IR) experiments are reported for insoluble monomolecular films of an equimolar mixture of 1,2-dipalmitoylphosphatidylcholine (DPPC) and 1,2-dipalmitoylphosphatidylserine (DPPS) at the A/W interface as a function of surface pressure and Ca2+ ion presence. The separate components showed a surface pressure-induced conformational ordering of the acyl chains. The conformational ordering occurred more cooperatively for the DPPS. Acyl chain perdeuteration of the DPPC permitted the observation of the response of the individual components in the binary mixture to changes in surface tension and to the presence of Ca2+. Plots of surface pressure versus CH2 or CD2 stretching frequencies were analyzed with a two-state model. At each surface pressure within the two-state region, the fraction of disordered form was the same for each lipid component, suggesting that they are well mixed on the surface. Calcium ion (5 mM in the subphase) produces almost no effect on the pressure-induced acyl chain ordering of the DPPC in a single component film, whereas the same levels of Ca2+ induce acyl chain ordering at all surface pressures in both components of the binary mixture. Thus, unlike the bulk phase mixture of DPPC/DPPS, the binary lipids in this mixed monolayer film appear to retain their miscibility in the presence of Ca2+. Finally, Ca(2+)-induced dehydration of the phosphate group was observed through characteristic frequency shifts in the asymmetric PO2- stretching mode.

Characterization of biological samples by two-dimensional infrared spectroscopy: Simulation of frequency, bandwidth, and intensity changes

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.

Location of Structural Transitions in an Isotopically Labeled Lung Surfactant SP-B Peptide by IRRAS

Pulmonary surfactant, a lipid/protein complex that lines the air/water interface in the mammalian lung, functions to reduce the work of breathing. Surfactant protein B (SP-B) is a small, hydrophobic protein that is an essential component of this mixture. Structure-function relationships of SP-B are currently under investigation as the protein and its peptide analogs are being incorporated into surfactant replacement therapies. Knowledge of the structure of SP-B and its related peptides in bulk and monolayer phases will facilitate the design of later generation therapeutic agents. Prior infrared reflection-absorption spectroscopic studies reported notable, reversible surface pressure-induced antiparallel beta-sheet formation in a synthetic peptide derived from human SP-B, residues 9-36 (SP-B(9-36)). In the current work, infrared reflection-absorption spectroscopy is applied in conjunction with isotopic labeling to detect the site and pressure dependence of the conformational change. SP-B(9-36), synthesized with (13)C=O-labeled Ala residues in positions 26, 28, 30, and 32, shifted the beta-sheet marker band to approximately 1600 cm(-1) and thus immediately identified this structural element within the labeled region. Surface pressure-induced alterations in the relative intensities of Amide I band constituents are interpreted using a semiempirical transition dipole coupling model. In addition, electron micrographs reveal the formation of tubular myelin structures from in vitro preparations using SP-B(9-36) in place of porcine SP-B indicating that the peptide has the potential to mimic this property of the native protein.

Interaction of recombinant surfactant protein D with lipopolysaccharide: conformation and orientation of bound protein by IRRAS and simulations.

Effective innate host defense requires early recognition of pathogens. Surfactant protein D (SP-D), shown to play a role in host defense, binds to the lipopolysaccharide (LPS) component of Gram-negative bacterial membranes. Binding takes place via the carbohydrate recognition domain (CRD) of SP-D. Recombinant trimeric neck+CRDs (NCRD) have proven valuable in biophysical studies of specific interactions. Although X-ray crystallography has provided atomic level information on NCRD binding to carbohydrates and other ligands, molecular level information about interactions between SP-D and biological ligands under physiologically relevant conditions is lacking. Infrared reflection-absorption spectroscopy (IRRAS) provides molecular structure information from films at the air/water interface where protein adsorption to LPS monolayers serves as a model for protein-lipid interaction. In the current studies, we examine the adsorption of NCRDs to Rd 1 LPS monolayers using surface pressure measurements and IRRAS. Measurements of surface pressure, Amide I band intensities, and LPS acyl chain conformational ordering, along with the introduction of EDTA, permit discrimination of Ca (2+)-mediated binding from nonspecific protein adsorption. The findings support the concept of specific binding between the CRD and heptoses in the core region of LPS. In addition, a novel simulation method that accurately predicts the IR Amide I contour from X-ray coordinates of NCRD SP-D is applied and coupled to quantitative IRRAS equations providing information on protein orientation. Marked differences in orientation are found when the NCRD binds to LPS compared to nonspecific adsorption. The geometry suggests that all three CRDs are simultaneously bound to LPS under conditions that support the Ca (2+)-mediated interaction.

A comparison of differential scanning calorimetric and Fourier transform infrared spectroscopic determination of mixing behavior in binary phospholipid systems

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.

Calorimetric and Fourier transform infrared spectroscopic studies on the interaction of glycophorin with phosphatidylserine/dipalmitoylphosphatidylcholine-d62 mixtures.

Glycophorin has been isolated in pure form from human erythrocyte membranes and reconstituted into lipid vesicles composed of binary mixtures of bovine brain phosphatidylserine (PS) and acyl-chain perdeuterated dipalmitoylphosphatidylcholine (DPPC-d62). The effect of protein on lipid melting behavior and order has been monitored with differential scanning calorimetry and Fourier transform infrared spectroscopy (FT-IR). The phase diagram for PS/DPPC-d62 is consistent with that previously reported for PS/DPPC (Stewart et al. (1979) Biochim. Biophys. Acta 556, 1-16) and indicates that acyl chain perdeuteration does not greatly alter the lipid mixing characteristics. The use of deuterated lipid allows the examination of lipid order by FT-IR of each lipid component in the binary mixtures as well as in the ternary (lipid/lipid/protein) systems. Addition of glycophorin to a 30:70 PS/DPPC-d62 binary lipid mixture results in a preferential glycophorin/PS interaction leading to bulk lipid enriched in DPPC-d62. This is revealed in two ways: first, through cooperative calorimetric transitions increased in temperature from the binary lipid system and second, through FT-IR melting curves of the DPPC-d62 component which shows transitions increased in both onset and completion temperatures in the presence of protein. In addition, non-cooperative melting events are observed at temperatures below the onset of phase separation. The FT-IR data are used to assign these non-cooperative events to the melting of the PS component. For the 50:50 lipid mixture with protein, two transitions are observed in the DSC experiments. The IR results indicate that both lipid components are involved with the lower temperature event.

Coupled External Reflectance FT-IR/Miniaturized Surface Film Apparatus for Biophysical Studies

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.