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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.

The effects of the hydrophobicity and the distribution of hydrophobic residues on the surfaces of some designed alpha-helical transmembrane peptides (acetyl-K2-L(m)-A(n)-K2-amide, where m + n = 24) on their solution behavior and interactions with phospholipids were examined. We find that although these peptides exhibit strong alpha-helix forming propensities in water, membrane-mimetic media, and lipid model membranes, the stability of the helices decreases as the Leu content decreases. Also, their binding to reversed phase high-performance liquid chromatography columns is largely determined by their hydrophobicity and generally decreases with decreases in the Leu/Ala ratio. However, the retention of these peptides by such columns is also affected by the distribution of hydrophobic residues on their helical surfaces, being further enhanced when peptide helical hydrophobic moments are increased by clustering hydrophobic residues on one side of the helix. This clustering of hydrophobic residues also increases peptide propensity for self-aggregation in aqueous media and enhances partitioning of the peptide into lipid bilayer membranes. We also find that the peptides LA3LA2 [acetyl-K2-(LAAALAA)3LAA-K2-amide] and particularly LA6 [acetyl-K2-(LAAAAAA)3LAA-K2-amide] associate less strongly with and perturb the thermotropic phase behavior of phosphatidylcholine bilayers much less than peptides with higher L/A ratios. These results are consistent with free energies calculated for the partitioning of these peptides between water and phospholipid bilayers, which suggest that LA3LA2 has an equal tendency to partition into water and into the hydrophobic core of phospholipid model membranes, whereas LA6 should strongly prefer the aqueous phase. We conclude that for alpha-helical peptides of this type, Leu/Ala ratios of greater than 7/17 are required for stable transmembrane associations with phospholipid bilayers.

Pulmonary surfactant, a thin lipid/protein film lining mammalian lungs, functions in vivo to reduce the work of breathing and to prevent alveolar collapse. Analogues of two hydrophobic surfactant proteins, SP-B and SP-C, have been incorporated into therapeutic agents for respiratory distress syndrome, a pathological condition resulting from deficiency in surfactant. To facilitate rational design of therapeutic agents, a molecular level understanding of lipid interaction with surfactant proteins or their analogues in aqueous monolayer films is necessary. The current work uses infrared reflection-absorption spectroscopy (IRRAS) to determine peptide conformation and the effects of S-palmitoylation on the lipid interactions of a synthetic 13 residue N-terminal peptide [SP-C13(palm)(2)] of SP-C, in mixtures with 1,2-dipalmitoylphosphatidylcholine (DPPC) or 1,2-dipalmitoylphosphatidylglycerol (DPPG). Two Amide I' features, at approximately 1655 and approximately 1639 cm(-1) in the peptide IRRAS spectra, are assigned to alpha-helical peptide bonds in hydrophobic and aqueous environments, respectively. In binary DPPC/SP-C13(palm)(2) films, the proportion of hydrated/hydrophobic helix increases reversibly with surface pressure (pi), suggestive of the peptide being squeezed out from hydrophobic regions of the monolayer. No such effect was observed for DPPG/peptide monolayers, indicative of stronger, probably electrostatic, interactions. Depalmitoylation produced a weakened interaction with either phospholipid as deduced from IRRAS spectra and from pi-area isotherms. S-Palmitoylation may modulate peptide hydration and conformation in the N-terminal region of SP-C and may thus permit the peptide to remain in the film at the high surface pressures present during lung compression. The unique capability of IRRAS to detect the surface pressure dependence of protein or peptide structure/interactions in a physiologically relevant model for surfactant is clearly demonstrated. Udgivelsesdato: 2002-Jul-2

The conformation and amide proton exchangeability of the peptide acetyl-K(2)-A(24)-K(2)-amide (A(24)) and its interaction with phosphatidylcholine bilayers were examined by a variety of physical techniques. When dissolved in or cast from methanol as a dried film, A(24) is predominantly alpha-helical. In aqueous media, however, A(24) exists primarily as a mixture of helical (though not necessarily alpha-helical) and random coiled structures, both of which allow rapid H-D exchange of all amide protons. When incorporated into phospholipids in the absence of water, A(24) also exists primarily as a transmembrane alpha-helix. However, upon hydration of that system, rapid exchange of all amide protons also occurs along with a marked change in the amide I absorption band of the peptide. Also, when dispersed with phosphatidylcholine in aqueous media, the conformation and thermal stability of A(24) are not significantly altered by the presence of the phospholipid or by its gel/liquid-crystalline phase transition. Differential scanning calorimetric and electron spin resonance spectroscopic studies indicate that A(24) has relatively minor effects on the thermodynamic properties of the lipid hydrocarbon chain-melting phase transition, that it does not abolish the lipid pretransition, and that its presence has no significant effect on the orientational order or rates of motion of the phospholipid hydrocarbon chains. We therefore conclude that A(24) has sufficient alpha-helical propensity, but insufficient hydrophobicity, to maintain a stable transmembrane association with phospholipid bilayers in the presence of water. Instead, it exists primarily as a dynamic mixture of helices and other conformers and resides mostly in the aqueous phase where it interacts weakly with the bilayer surface or with the polar/apolar interfacial region of phosphatidylcholine bilayers. Thus, polyalanine-based peptides are not good models for the transmembrane alpha-helical segments of natural membrane proteins.

We have utilized Fourier transform infrared spectroscopy to study the interaction of the antimicrobial peptide gramicidin S (GS) with lipid micelles and with lipid monolayer and bilayer membranes as a function of temperature and of the phase state of the lipid. Since the conformation of GS does not change under the experimental conditions employed in this study, we could utilize the dependence of the frequency of the amide I band of the central beta-sheet region of this peptide on the polarity and hydrogen-bonding potential of its environment to probe GS interaction with and location in these lipid model membrane systems. We find that the GS is completely or partially excluded from the gel states of all of the lipid bilayers examined in this study but strongly partitions into lipid micelles, monolayers, or bilayers in the liquid-crystalline state. Moreover, in general, the penetration of GS into zwitterionic and uncharged lipid bilayer coincides closely with the gel to liquid-crystalline phase transition of the lipid. However, GS begins to penetrate into the gel-state bilayers of anionic phospholipids prior to the actual chain-melting phase transition, while in cationic lipid bilayers, GS does not partition strongly into the liquid-crystalline bilayer until temperatures well above the chain-melting phase transition are reached. In the liquid-crystalline state, the polarity of the environment of GS indicates that this peptide is located primarily at the polar/apolar interfacial region of the bilayer near the glycerol backbone region of the lipid molecule. However, the depth of GS penetration into this interfacial region can vary somewhat depending on the structure and charge of the lipid molecule. In general, GS associates most strongly with and penetrates most deeply into more disordered bilayers with a negative surface charge, although the detailed chemical structure of the lipid molecule and physical organization of the lipid aggregate (micelle versus monolayer versus bilayer) also have minor effects on these processes.

Fatty acid-binding proteins (FABPs) found in many tissues constitute a family of low molecular weight proteins that are suggested to function as intracellular transporters of fatty acids. Studies of the transfer kinetics of fluorescent anthroyloxy-labeled long-chain fatty acids from FABP to model membranes led to the suggestion that the FABPs, typically considered to be cytosolic proteins, could nevertheless interact directly with membranes [Wootan, M. G., et al. (1993) Biochemistry 32, 8622-8627]. In the current study, the interaction of the adipocyte FABP (A-FABP) with vesicles of various phospholipids has been directly measured and confirmed with FTIR spectroscopy. The strength of this interaction was inferred from the lowering of the gel-liquid-crystal phase transition temperature as monitored from temperature-induced variations in the acyl chain CH2 stretching frequencies and from the intensities of the components of the CH2 wagging progressions. A-FABP interacts more strongly with anionic phospholipids (phosphatidylserine and cardiolipin) than with zwitterionic phosphatidylcholine. Unsaturation in the acyl chains leads to a greater reduction in Tm (stronger lipid-protein interaction). In contrast, neutralization of A-FABP surface charges by acetylation considerably weakens the interaction. Comparison of the shifts in lipid melting temperatures with those induced by other proteins suggests that A-FABP behaves like a typical peripheral membrane protein. The degree of membrane interaction correlates directly with the rate of fatty acid transfer, suggesting that contact between A-FABP and membranes is functionally related to its fatty acid transport properties. As expected, the protein exhibits a predominantly beta-sheet structure. It was found to aggregate with increasing temperature. With the exception of minor differences between the pure and lipid-associated A-FABP in the 1640-1660 cm-1 region, both the protein structure and thermal stability appeared essentially unchanged upon interaction with the lipid.

Acyl chain perdeuterated dimyristoylphosphatidylcholine (DMPC-d54) and dimyristoyphosphatidylserine (DMPS-d54) were incorporated into human erythrocytes. Light microscopy demonstrated that erythrocytes incubated with an equimolar mixture of DMPC-d54/DMPS or DMPC/DMPS-d54 remained mostly discocytic whereas cells incubated with either DMPC-d54 or DMPS-d54 alone became echinocytic or stomatocytic, respectively. Cells in which the aminophospholipid translocating protein was inhibited became echinocytic when incubated with DMPS-d54. Fourier transform infrared (FTIR) spectroscopy was used to monitor conformational order in the acyl chains of the incorporated phospholipid, as detected through the asymmetric CD2 stretching vibrations in the intact cells. In cells incubated with equimolar mixtures of DMPC-d54/DMPS or DMPC/DMPS-d54, the deuterated species exhibited no thermotropic phase transitions but revealed chain order intermediate between the gel and liquid-crystal states. In contrast, DMPS-d54 incorporated into the outer leaflet of echinocytic erythrocytes was conformationally ordered while the same species incorporated into the inner leaflet of stomatocytic erythrocytes was highly disordered at all temperatures studied. Finally, DMPC-d54 incorporated into the outer leaflet of echinocytic erythrocytes exhibited a phase transition, suggesting that this species persists in domains. These data indicate that the acyl chain conformational order of specific phospholipids in the intact human erythrocyte is changed with alterations in cell morphology.

Oligonucleotide-directed site-specific mutagenesis was carried out on pyruvate decarboxylase (EC 4.1.1.1) from Saccharomyces cerevisiae at three of the four cysteines (152, 221, and 222), the fourth (69) being buried according to X-ray crystallographic results [Arjunan et al. (1996) J. Mol. Biol. 256, 590-600]. All of the variants still retained significant activity, and all could be purified to homogeneity. FT-IR experiments were run on the C221S, C222S, C221S/C222S and C152A variants, as well as on the wild-type enzyme. There is a band present at 2557 cm-1 in the spectra of all variants and the wild-type enzyme, except in the spectrum of the C152A variant. This frequency is appropriate to a cysteine S-H stretching mode. It was therefore concluded that C152 is the only undissociated cysteine on the enzyme at pH 6.0, the pH optimum of this enzyme, whereas C221, C222, and C69 are all ionized. Isoelectric focusing experiments were carried out on all of these variants, as well as on the H92A variant (H92 is across the domain divide on the alpha domain, from C221 located on the beta domain). The variation in isoelectric points deduced from the data was consistent with removal of negative charges concomitant with the C221S, C222S, and C221S/C222S substitutions and removal of a positive charge with the H92A substitution when compared to that of the wild-type enzyme. The results of these two types of experiments are in good accord and suggest that the site of substrate activation at C221 [Baburina et al. (1994) Biochemistry 33, 5630-5635] is comprised of a Cys221S- +HHis92 ion pair, not unlike that found in papain and glyceraldehyde-3-phosphate dehydrogenase. This finding suggests that the regulatory site of this enzyme has been optimized for nucleophilic reactivity between the thiolate of C221 and the keto carbon of the 2-oxoacid.

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)

Acholeplasma laidlawii B has been grown highly enriched in myristic, pentadecanoic, and palmitic acids. The conformational order in the acyl chains of living cell membranes has been compared with that in the membranes of lysed cells and the lipids extracted therefrom, using Fourier-transform infrared (FT-IR) spectroscopic techniques. A gel-liquid-crystal-phase transition (25-45 degrees C) was noted for both the live cells and the membranes. Surprisingly, CH2 wagging progressions, characteristic of coupled oscillators from acyl chains in the all-trans conformation, were detected in the live cells and in the cell membrane. A simple model suggested the presence of 1.7 gauche bonds/chain at the growth temperature (37 degrees C) in each case. Conformational order in the live cells and in the membranes was virtually identical over the range of cell viability (5-40 degrees C), as measured by the thermotropic responses of the k = 1 and k = 2 components of the wagging mode progression. This result was confirmed by studies of the thermotropic response of the symmetric CH2 stretching vibrations, a qualitative index of acyl chain order. In contrast, the membrane lipid extracts (i) show much more conformational disorder from 5 to 25 degrees C than either the live cells or the membranes, (ii) undergo a gel-liquid transition over a broader temperature range and with a reduced magnitude of change in the symmetric CH2 stretching frequency, and (iii) demonstrate a second transition centered at 50 degrees C, which is detected by a large increase in the localized CH2 wagging mode (1368 cm-1) that arises from conformationally disordered kink+gtg states.(ABSTRACT TRUNCATED AT 250 WORDS)

CaATPase from rabbit sarcoplasmic reticulum (SR) has been isolated, purified, and reconstituted into unilamellar vesicles with 1,2-dipentadecanoylphosphatidylcholine (DPePC) and acyl-chain-perdeuterated 1,2-dipalmitoylphosphatidylcholine (DPPC-d62). For total lipid:protein mole ratios between 25 and 70, a constant "hard-to-exchange" phospholipid population (HEPP) of 12 +/- 4 native phospholipid molecules per protein monomer is observed, consistent with the studies of Bick et al. [(1991) Arch. Biochem. Biophs. 286, 346-352]. Thermotropic and conformational properties of the lipids in native SR and in the reconstituted systems were probed with FT-IR spectroscopy. The native SR phospholipids undergo a broad phase transition centered at about 1-2 degrees C and are thus disordered under physiological conditions. The thermotropic behavior of CH2 wagging progressions characteristic of the palmitate chains differs from that of the total lipid population and is suggestive of membrane microheterogeneity. The individual thermotropic and conformational properties of the HEPP and the exogenous lipid in reconstituted vesicles containing CaATPase and DPPC-d62 were monitored. At temperatures below the onset of the gel-liquid-crystal phase transition, the HEPP possesses significant conformational disorder, and exhibits the monotonic introduction of gauche rotamers as the temperature is raised from -55 to 27 degrees C, in contrast to the exogenous lipid, which exhibits a constant high order over the same temperature range. Nevertheless, the HEPP undergoes a residual order-disorder phase transition with similar but not identical parameters (half-width, midpoint temperature) to the gel-liquid-crystal transition of the bulk (exogenous) lipid in the reconstituted systems.(ABSTRACT TRUNCATED AT 250 WORDS)

The structure and dynamics of synthetic melittin (MLT) and MLT analogues bound to monomyristoylphosphatidylcholine micelles, dimyristoylphosphatidylcholine vesicles, and diacylphosphatidylcholine films have been investigated by fluorescence, CD, attenuated total reflectance (ATR) FTIR, and 13C NMR spectroscopy. All of these methods provide information about peptide secondary structure and/or about the environment of the single tryptophan side chain in these lipid environments. ATR-FTIR data provide additional information about the orientation of helical peptide segments with respect to the bilayer plane. Steady-state fluorescence anisotropy, fluorescence lifetime, and 13C NMR relaxation data are used in concert to provide quantitative information about the dynamics of a single 13C-labeled tryptophan side chain at position 19 in lipid-bound MLT, and at positions 17, 11, and 9, respectively, in lipid-bound MLT analogues. Peptide chain dynamics are probed by NMR relaxation studies of 13C alpha-labeled glycine incorporated into each of the MLT peptides at position 12. The cumulative structural and dynamic data are consistent with a model wherein the N-terminal alpha-helical segment of these peptides is oriented perpendicular to the bilayer plane. Correlation times for the lysolipid-peptide complexes provide evidence for binding of a single peptide monomer per micelle. A model for the membranolytic action of MLT and MLT-like peptides is proposed.

KLLLLKLLLLKLLLLKLLLLK (KL(4)) has been suggested to mimic some aspects of the pulmonary surfactant protein SP-B and has been tested clinically as a therapeutic agent for respiratory distress syndrome in premature infants [Cochrane, C. G., and Revak, S. D. (1991) Science 254, 566-568]. It is of obvious interest to understand the mechanism of KL(4) function as a guide for design of improved therapeutic agents. Attenuated total reflection (ATR) IR measurements have indicated that KL(4) is predominantly alpha-helical with a transmembrane orientation in lipid multilayers (1), a geometry quite different from the originally proposed peripheral membrane lipid interaction. However, the lipid multilayer model required for ATR may not be the best experimental paradigm to mimic the in vivo function of KL(4). In the current experiments, IR reflection-absorption spectroscopy (IRRAS) was used to evaluate peptide secondary structure in monolayers at the air/water interface, the physical state that best approximates the alveolar lining. In contrast to the ATR-IR results, KL(4) (2.5-5 mol %) films with either DPPC or DPPC/DPPG (7/3 mol ratio) adopted an antiparallel beta-sheet structure at all surface pressures studiedor =5 mN/m, including pressures physiologically relevant for lung function (40-72 mN/m). In contrast, in DPPG/KL(4) films, the dominant conformation was the alpha-helix over the entire pressure range, a possible consequence of enhanced electrostatic interactions. IRRAS has thus provided unique molecular structure information and insight into KL(4)/lipid interaction in a physiologically relevant state. A structural model is proposed for the response of the peptide to surface pressure changes.

Surfactant protein A (SP-A), the most abundant pulmonary surfactant protein, is implicated in multiple biological functions including surfactant homeostasis, biophysical activity, and host defense. SP-A forms ternary complexes with lipids and Ca2+ which are important for protein function. The current study uses infrared (IR) transmission spectroscopy to investigate the bulk-phase interaction between SP-A, 1,2-dipalmitoylphosphatidylcholine (DPPC), and Ca2+ ions along with IR reflection-absorption spectroscopy (IRRAS) to examine protein secondary structure and lipid orientational order in monolayer films in situ at the air/water interface. The amide I contour of SP-A reveals two features at 1653 and 1636 cm(-1) arising from the collagen-like domain and a broad feature at 1645 cm(-1) suggested to arise from the carbohydrate recognition domain (CRD). SP-A secondary structure is unchanged in lipid monolayers. Thermal denaturation of SP-A in the presence of either DPPC or Ca2+ ion reveals a sequence of events involving the initial melting of the collagen-like region, followed by formation of intermolecular extended forms. Interestingly, these spectral changes were inhibited in the ternary system, showing that the combined presence of both DPPC and Ca2+ confers a remarkable thermal stability upon SP-A. The ternary interaction was revealed by the enhanced intensity of the asymmetric carboxylate stretching vibration. The IRRAS measurements indicated that incorporation of SP-A into preformed DPPC monolayers at a surface pressure of 10 mN/m induced a decrease in the average acyl chain tilt angle from 35 degrees to 28 degrees. In contrast, little change in chain tilt was observed at surface pressures of 25 or 40 mN/m. These results are consistent with and extend the fluorescence microscopy studies of Keough and co-workers [Ruano, M. L. F., et al. (1998) Biophys. J. 74, 1101-1109] in which SP-A was suggested to accumulate at the liquid-expanded/liquid-condensed boundary. Overall these experiments reveal the remarkable stability of SP-A in diverse, biologically relevant environments.

Intestinal fatty acid binding protein (IFABP) appears to interact directly with membranes during fatty acid transfer [Hsu, K. T., and Storch, J. (1996) J. Biol. Chem. 271, 13317-13323]. The largely alpha-helical "portal" domain of IFABP was critical for these protein--membrane interactions. In the present studies, the binding of IFABP and a helixless variant of IFABP (IFABP-HL) to acidic monolayers of 1,2-dimyristoylphosphatidic acid (DMPA) has been monitored by surface pressure measurements, Brewster angle microscopy (BAM), and infrared reflection-absorption spectroscopy (IRRAS). Protein adsorption to DMPA exhibited a two phase kinetic process consisting of an initial slow phase, arising from protein binding to the monolayer and/or direct interfacial adsorption, and a more rapid phase that parallels formation of lipid-containing domains. IFABP exhibited more rapid changes in both phases than IFABP-HL. The second phase was absent when IFABP interacted with zwitterionic monolayers of 1,2-dipalmitoylphosphatidylcholine, revealing the important role of electrostatics at this stage. BAM images of DMPA monolayers with either protein revealed the formation of domains leading eventually to rigid films. Domains of DMPA/IFABP-HL formed more slowly and were less rigid than with the wild-type protein. Overall, the IRRAS studies revealed a protein-induced conformational ordering of the lipid acyl chains with a substantially stronger ordering effect induced by IFABP. The physical measurements thus suggested differing degrees of direct interaction between the proteins and DMPA monolayers with the IFABP/DMPA interaction being somewhat stronger. These data provide a molecular structure rationale for previous kinetic measurements indicating that the helical domain is essential for a collision-based mechanism of fatty acid transfer to phospholipid membranes [Corsico, B., Cistola, D. P., Frieden, C. and Storch, J. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 12174-12178].

Acyl chain perdeuterated dimyristoylphosphatidylcholine (DMPC-d54) and dimyristoylphosphatidylserine (DMPS-d54) were incorporated by incubation into human erythrocytes. Light microscopic analysis demonstrated that erythrocytes incubated with DMPC-d54 became echinocytic while those incubated with DMPS-d54 became stomatocytic. This indicates that DMPC-d54 was incorporated preferentially into the outer monolayer whereas DMPS-d54 was selectively incorporated into the inner monolayer. Fourier transform infrared (FTIR) spectroscopy was used to monitor the conformational order of the incorporated phospholipids. The asymmetric CD2 stretching frequency of the inserted perdeuterated acyl chains was measured in both isolated membranes and intact erythrocytes as a function of temperature. DMPC-d54 incorporated into erythrocytes exhibited a cooperative phase transition at approximately 19 degrees C, i.e., at the same temperature as pure vesicles. In contrast, DMPS-d54 incorporated into red cells exhibited no phase transition, but possessed conformational order similar to that of the liquid-crystalline state. These results suggest that DMPC-d54 persists in domains in the outer monolayer while DMPS-d54 is dispersed in the inner monolayer. These experiments are the first to demonstrate that FTIR spectroscopy can be utilized to monitor directly a specific species of lipid molecule from the entire phospholipid population.

SP-C, a highly hydrophobic, 3.7-kDa protein constituent of lung surfactant, has been isolated from bovine lung lavage, purified, and reconstituted into binary lipid mixtures of 1,2-dipalmitoyl-phosphatidylcholine (DPPC) and 1,2-dipalmitoylphosphatidylglycerol (DPPG). Fourier transform infrared (FT-IR) spectroscopy has been applied to examine SP-C secondary structure, the average orientation of alpha-helical segments relative to the bilayer normal in membrane films, and the effect of protein on the thermotropic properties of the phospholipid acyl chains. In addition, dynamic surface measurements were made on phospholipid films at the A/W interface in the presence and absence of SP-C. SP-C (0.5 mol %) was found to possess about 60% alpha-helical secondary structure in lipid vesicles. Higher levels (1.5 mol %) of SP-C resulted in a slight increase of beta-forms, possibly resulting from protein aggregation. The helical segments exhibited an average angle of orientation of about 24 degrees with respect to the bilayer normal, suggesting a trans-bilayer orientation of the peptide. The observation that 70% of the peptide bond hydrogens are hard to exchange in D2O further reflects the hydrophobic nature of the molecule. SP-C produced little effect on the thermotropic properties of the binary lipid mixture, as measured from acyl chain C-H and C-D stretching frequencies. However, the presence of 1 mol % protein markedly reduced the viscance and increased the elasticity of surface films suggesting a mechanism by which SP-C facilitates the spreading of phospholipids on an aqueous surface. The possible physiological consequences of these observations are discussed.

The use of CD2 rocking modes in the IR spectrum as quantitative probes of phospholipid conformational disorder has recently been described for aqueous dispersions of 1,2-dipalmitoylphosphatidylcholine (DPPC) and DPPC/cholesterol mixtures [Mendelsohn et al. (1989) Biochemistry 28, 8934-8939; Davies et al. (1990) Biochemistry 29, 4368-4373]. Initial studies focused at the 4, 6, and 10 acyl chain positions of DPPC. In the current work, the method is extended to the 2, 3, 12, and 13 positions. Conformational disorder in the L alpha phase is approximately the same (about 20% gauche) at positions 4, 10, and 13, but an unexpected higher value is observed (about 30%) at the 6 position. Cholesterol (33 mol%) restricts gauche rotamer formation by factors ranging from 6 to 9 at positions 4 and 6, respectively, to 1.5-2 at positions 10, 12, and 13. Quantitative analysis for the DPPC/cholesterol "liquid-ordered" phase indicates the occurrence of 1.2 gauche bonds/chain, a marked reduction from the 3.6-4.2 gauche bonds/chain for DPPC alone. Proximity to the ester moiety at acyl chain position 3 perturbs the vibrational coupling patterns of the CD2 rocking modes and eliminates their sensitivity to conformational change. In addition, the feasibility of a method based on the conformation-dependent coupling between CD2 rocking frequencies of two successive CD2 groups for the quantitative detection of specific, position-dependent king (gtg') and isolated gauche (gtt) conformers is demonstrated. Finally, comparisons between IR measurements and explicit theoretical predictions of acyl chain conformational order are presented.

A method originally proposed by Snyder and Poore [(1973) Macromolecules 6, 708-715] as a specific probe of trans-gauche isomerization in hydrocarbon chains and recently applied [Mendelsohn et al. (1989) Biochemistry 28, 8934-8939] to the quantitative determination of phospholipid acyl chain conformational order is utilized to monitor the effects of cholesterol at various depths in dipalmitoylphosphatidylcholine (DPPC) bilayers. The method is based on the observation that the CD2 rocking modes from the acyl chains of specifically deuterated phospholipids occur at frequencies in the Fourier transform infrared spectrum which depend upon the local geometry (trans or gauche) of the C-C-C skeleton surrounding a central CD2 group. Three specifically deuterated derivatives of DPPC, namely, 4,4,4',4'-d4 DPPC (4-d4 DPPC), 6,6,6',6'-d4 DPPC (6-d4 DPPC), and 12,12,12',12'-d4 DPPC (12-d4 DPPC), have been synthesized, and the effects of cholesterol addition at 2:1 DPPC/cholesterol (mol:mol) on acyl chain order at various temperatures have been determined. At 48 degrees C, cholesterol inhibits gauche rotamer formation by factors of approximately 9 and approximately 6 at positions 6 and 4, respectively, of the acyl chains, thus demonstrating a strong ordering effect in regions of the bilayer where the sterol rings are presumed to insert parallel to the DPPC acyl chains. In contrast, the ability of the sterol to order the acyl chains is much reduced at the 12-position. The sterol demonstrates only a slight disordering of phospholipid gel phases. Finally, the contributions of different classes of gauche conformers to the spectra have been determined.(ABSTRACT TRUNCATED AT 250 WORDS)