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.     

Secondary structure and lipid interactions of the N-terminal segment of pulmonary surfactant SP-C in Langmuir films: IR reflection-absorption spectroscopy and surface pressure studies

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.     

An infrared reflection-absorption spectroscopy study of the secondary structure in (KL4)4K,a therapeutic agent for respiratory distress syndrome,in aqueous monolayers with phospholipids

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 studied > or =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.     

External reflection absorption infrared spectroscopy study of lung surfactant proteins SP-B and SP-C in phospholipid monolayers at the air/water interface.

The interactions of the hydrophobic pulmonary surfactant proteins SP-B and SP-C with 1,2-dipalmitoylphosphatidylcholine in mixed, spread monolayer films have been studied in situ at the air/water interface with the technique of external reflection absorption infrared spectroscopy (IRRAS). SP-C has a mostly alpha-helical secondary structure both in the pure state and in the presence of lipids, whereas SP-B secondary structure is a mixture of alpha-helical and disordered forms. When films of SP-B/1,2-dipalmitoylphosphatidylcholine are compressed to surface pressures (pi) greater than approximately 40-43 mN/m, the protein is partially (15-35%) excluded from the surface, as measured by intensity ratios of the peptide bond amide l/lipid C==O stretching vibrations. The extent of exclusion increases as the protein/lipid ratio in the film increases. In contrast, SP-C either remains at the surface at high pressures or leaves accompanied by lipids. The amide l peak of SP-C becomes asymmetric as a result of the formation of intermolecular sheet structures (1615-1630 cm-1) suggestive of peptide aggregation. The power of the IRRAS experiment for determination of film composition and molecular structure, i.e., as a direct test of the squeeze-out hypothesis of pulmonary surfactant function, is evident from this work.     

Structure and orientation of lung surfactant SP-C and L-alpha-dipalmitoylphosphatidylcholine in aqueous monolayers.

SP-C, a pulmonary surfactant-specific protein, aids the spreading of the main surfactant phospholipid L-alpha-dipalmitoylphosphatidylcholine (DPPC) across air/water interfaces, a process that has possible implications for in vivo function. To understand the molecular mechanism of this process, we have used external infrared reflection-absorption spectroscopy (IRRAS) to determine DPPC acyl chain conformation and orientation as well as SP-C secondary structure and helix tilt angle in mixed DPPC/SP-C monolayers in situ at the air/water interface. The SP-C helix tilt angle changed from approximately 24 degrees to the interface normal in lipid bilayers to approximately 70 degrees in the mixed monolayer films, whereas the acyl chain tilt angle of DPPC decreased from approximately 26 degrees in pure lipid monolayers (comparable to bilayers) to approximately 10 degrees in the mixed monolayer films. The protein acts as a "hydrophobic lever" by maximizing its interactions with the lipid acyl chains while simultaneously permitting the lipids to remain conformationally ordered. In addition to providing a reasonable molecular mechanism for protein-aided spreading of ordered lipids, these measurements constitute the first quantitative determination of SP-C orientation in Langmuir films, a paradigm widely used to simulate processes at the air/alveolar interface.     

Monolayer–multilayer transitions in a lung surfactant model: IR reflection–absorption spectroscopy and atomic force microscopy

A hydrophobic pulmonary surfactant protein, SP-C, has been implicated in surface-associated activities thought to facilitate the work of breathing. Model surfactant films composed of lipids and SP-C display a reversible transition from a monolayer to surface-associated multilayers upon compression and expansion at the air/water (A/W) interface. The molecular-level mechanics of this process are not yet fully understood. The current work uses atomic force microscopy on Langmuir–Blodgett films to verify the formation of multilayers in a dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, cholesterol, and SP-C model system. Isotherms of SP-C-containing films are consistent with exclusion and essentially complete respreading during compression and expansion, respectively. Multilayer formation was not detected in the absence of SP-C. Most notable are the results from IR reflection–absorption spectroscopy (IRRAS) conducted at the A/W interface, where the position and intensity of the Amide I band of SP-C reveal that the predominantly helical structure changes its orientation in monolayers versus multilayers. IRRAS measurements indicate that the helix tilt angle changed from approximately 80° in monolayers to a transmembrane orientation in multilayers. The results constitute the first quantitative measure of helix orientation in mixed monolayer/multilamellar domains at the A/W interface and provide insight into the molecular mechanism for SP-C-facilitated respreading of surfactant.     

Thermal stability and DPPC/Ca2+ interactions of pulmonary surfactant SP-A from bulk-phase and monolayer IR spectroscopy.

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.     

Folding of β-barrel membrane proteins in lipid bilayers — Unassisted and assisted folding and insertion

In cells, β-barrel membrane proteins are transported in unfolded form to an outer membrane into which they fold and insert. Model systems have been established to investigate the mechanisms of insertion and folding of these versatile proteins into detergent micelles, lipid bilayers and even synthetic amphipathic polymers. In these experiments, insertion into lipid membranes is initiated from unfolded forms that do not display residual β-sheet secondary structure. These studies therefore have allowed the investigation of membrane protein folding and insertion in great detail. Folding of β-barrel membrane proteins into lipid bilayers has been monitored from unfolded forms by dilution of chaotropic denaturants that keep the protein unfolded as well as from unfolded forms present in complexes with molecular chaperones from cells. This review is aimed to provide an overview of the principles and mechanisms observed for the folding of β-barrel transmembrane proteins into lipid bilayers, the importance of lipid–protein interactions and the function of molecular chaperones and folding assistants. This article is part of a Special Issue entitled: Lipid–protein interactions.     

Adsorption of GST-PI3Kγ at the Air-Buffer Interface and at Substrate and Nonsubstrate Phospholipid Monolayers

The recruitment of phosphoinositide 3-kinase γ (PI3Kγ) to the cell membrane is a crucial requirement for the initiation of inflammation cascades by second-messenger production. In addition to identifying other regulation pathways, it has been found that PI3Kγ is able to bind phospholipids directly. In this study, the adsorption behavior of glutathione S-transferase (GST)-PI3Kγ to nonsubstrate model phospholipids, as well as to commercially available substrate inositol phospholipids (phosphoinositides), was investigated by use of infrared reflection-absorption spectroscopy (IRRAS). The nonsubstrate phospholipid monolayers also yielded important information about structural requirements for protein adsorption. The enzyme did not interact with condensed zwitterionic or anionic monolayers; however, it could penetrate into uncompressed fluid monolayers. Compression to values above its equilibrium pressure led to a squeezing out and desorption of the protein. Protein affinity for the monolayer surface increased considerably when the lipid had an anionic headgroup and contained an arachidonoyl fatty acyl chain in sn-2 position. Similar results on a much higher level were observed with substrate phosphoinositides. No structural response of GST-PI3Kγ to lipid interaction was detected by IRRAS. On the other hand, protein adsorption caused a condensing effect in phosphoinositide monolayers. In addition, the protein reduced the charge density at the interface probably by shifting the pK values of the phosphate groups attached to the inositol headgroups. Because of their strongly polar headgroups, an interaction of the inositides with the water molecules of the subphase can be expected. This interaction is disturbed by protein adsorption, causing the ionization state of the phosphates to change.     

In Vitro Reconstitution of the Initial Stages of the Bacterial Cell Division Machinery

Fission of many prokaryotes as well as some eukaryotic organelles depends on the self-assembly of the FtsZ protein into a membrane-associated ring structure early in the division process. Different components of the machinery are then sequentially recruited. Although the assembly order has been established, the molecular interactions and the understanding of the force-generating mechanism of this dividing machinery have remained elusive. It is desirable to develop simple reconstituted systems that attempt to reproduce, at least partially, some of the stages of the process. High-resolution studies of Escherichia coli FtsZ filaments’ structure and dynamics on mica have allowed the identification of relevant interactions between filaments that suggest a mechanism by which the polymers could generate force on the membrane. Reconstituting the membrane-anchoring protein ZipA on E. coli lipid membrane on surfaces is now providing information on how the membrane attachment regulates FtsZ polymer dynamics and indicates the important role played by the lipid composition of the membrane.     

Deletion of the helical motif in the intestinal fatty acid-binding protein reduces its interactions with membrane monolayers: Brewster angle microscopy, IR reflection-absorption spectroscopy, and surface pressure studies

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].     

Advances in the characterization of supported lipid films with the atomic force microscope

During the past decade, the atomic force microscope (AFM) has become a key technique in biochemistry and biophysics to characterize supported lipid films, as testified by the continuous growth in the number of papers published in the field. The unique capabilities of AFM are: (i) capacity to probe, in real time and in aqueous environment, the surface structure of lipid films; (ii) ability to directly measure physical properties at high spatial resolution; (iii) possibility to modify the film structure and biophysical processes in a controlled way. Such experiments, published up to June 2000, are the focus of the present review. First, we provide a general introduction on the preparation and characterization of supported lipid films as well as on the principles of AFM. The section 'Structural properties' focuses on the various applications of AFM for characterizing the structure of supported lipid films: visualization of molecular structure, formation of structural defects, effect of external agents, formation of supported films, organization of phase-separated films (coexistence region, mixed films) and, finally, the use of supported lipid bilayers for anchoring biomolecules such as DNA, enzymes and crystalline protein arrays. The section 'Physical properties' introduces the principles of force measurements by AFM, interpretation of these measurements and their recent application to supported lipid films and related structures. Finally, we highlight the major achievements brought by the technique and some of the current limitations.     

Domain structure and molecular conformation in annexin V/1,2-dimyristoyl-sn-glycero-3-phosphate/Ca2+ aqueous monolayers: a Brewster angle microscopy/infrared reflection-absorption spectroscopy study.

Annexins comprise a family of proteins that exhibit a Ca2+-dependent binding to phospholipid membranes that is possibly relevant to their in vivo function. Although substantial structural information about the ternary (protein/lipid/Ca2+) interaction in bulk phases has been derived from a variety of techniques, little is known about the temporal and spatial organization of ternary monolayer films. The effect of Ca2+ on the interactions between annexin V (AxV) and anionic DMPA monolayers was therefore investigated using three complementary approaches: surface pressure measurements, infrared reflection-absorption spectroscopy (IRRAS), and Brewster angle microscopy (BAM). In the absence of Ca2+, the injection of AxV into an aqueous subphase beneath a DMPA monolayer initially in a liquid expanded phase produced BAM images revealing domains of protein presumably surrounded by liquid-expanded lipid. The protein-rich areas expanded with time, resulting in reduction of the area available to the DMPA and, eventually, in the formation of condensed lipid domains in spatial regions separate from the protein film. There was thus no evidence for a specific binary AxV/lipid interaction. In contrast, injection of AxV/Ca2+ at a total Ca2+ concentration of 10 microM beneath a DMPA monolayer revealed no pure protein domains, but rather the slow formation of pinhead structures. This was followed by slow (>2 h) rigidification of the whole film accompanied by an increase in surface pressure, and connection of solid domains to form a structure resembling strings of pearls. These changes were characteristic of this specific ternary interaction. Acyl chain conformational order of the DMPA, as measured by nu(sym)CH2 near 2850 cm(-1), was increased in both the AxV/DMPA and AxV/DMPA/Ca2+ monolayers compared to either DMPA monolayers alone or in the presence of Ca2+. The utility of the combined structural and temporal information derived from these three complementary techniques for the study of monolayers in situ at the air/water interface is evident from this work.     

Scanning Force Microscopy at the Air-Water Interface of an Air Bubble Coated with Pulmonary Surfactant

To study the structure-function relationship of pulmonary surfactant under conditions close to nature, molecular films of a model system consisting of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, and surfactant-associated protein C were prepared at the air-water interface of air bubbles about the size of human alveoli (diameter of 100 μm). The high mechanical stability as well as the absence of substantial film flow, inherent to small air bubbles, allowed for scanning force microscopy (SFM) directly at the air-water interface. The SFM topographical structure was correlated to the local distribution of fluorescent-labeled dipalmitoylphosphatidylcholine, as revealed from fluorescence light microscopy of the same bubbles. Although SFM has proven before to be exceptionally well suited to probe the structure of molecular films of pulmonary surfactant, the films so far had to be transferred onto a solid support from the air-water interface of a film balance, where they had been formed. This made them prone to artifacts imposed by the transfer. Moreover, the supported monolayers disallowed the direct observation of the structural dynamics associated with expansion and compression of the films as upon breathing. The current findings are compared in this respect to our earlier findings from films, transferred onto a solid support.     

Genotype-specific differences in structural features of hepatitis C virus (HCV) p7 membrane protein

The 63 amino acid polytopic membrane protein, p7, encoded by hepatitis C virus (HCV) is involved in the modulation of electrochemical gradients across membranes within infected cells. Structural information relating to p7 from multiple genotypes has been generated in silico (e.g. genotype (GT) 1a), as well as obtained from experiments in form of monomeric and hexameric structures (GTs 1b and 5a, respectively). However, sequence diversity and structural differences mean that comparison of their channel gating behaviour has not thus far been simulated. Here, a molecular model of the monomeric GT 1a protein is optimized and assembled into a hexameric bundle for comparison with both the 5a hexamer structure and another hexameric bundle generated using the GT 1b monomer structure. All bundles tend to turn into a compact structure during molecular dynamics (MD) simulations (Gromos96 (ffG45a3)) in hydrated lipid bilayers, as well as when simulated at ‘low pH’, which may trigger channel opening according to some functional studies. Both GT 1a and 1b channel models are gated via movement of the parallel aligned helices, yet the scenario for the GT 5a protein is more complex, with a short N-terminal helix being involved. However, all bundles display pulsatile dynamics identified by monitoring water dynamics within the pore.     

Secondary structure in lung surfactant SP-B peptides: IR and CD studies of bulk and monolayer phases

Pulmonary surfactant protein SP-B is known to facilitate adsorption and spreading of surfactant components across the air/water interface. This property appears essential for in vivo function in the alveolar subphase and at the air/alveolar surface. Three peptides with amino acid sequences based on SP-B containing predicted alpha-helical regions (SP-B(1--20), SP-B(9--36A), SP-B(40--60A)) have been synthesized to probe structure-function relationships and protein-lipid interaction in bulk phase and monolayer environments. IR and CD studies are reported along with traditional surface pressure-molecular area (pi-A) isotherms and IR reflection-absorption spectroscopy (IRRAS) investigations conducted at the air/water interface. In bulk phase, helix-promoting environments (methanol and aqueous dispersions of lipid vesicles), SP-B(1--20) and SP-B(9--36A) contained significant amounts of alpha-helical structure, whereas varying degrees of alpha-helix, random coil, and beta-sheet were observed in aqueous solutions and monolayers. The most striking behavior was observed for SP-B(9--36A), which displayed reversible surface pressure-induced beta-sheet formation. Bulk phase lipid melting curves and monolayer experiments with peptide-lipid mixtures showed subtle differences in the degree of bulk phase interaction and substantial differences in peptide surface activity. The uniqueness of IRRAS is emphasized as the importance of evaluating secondary structure in both bulk phase and monolayer environments for lung surfactant peptide mimics is demonstrated.     

Investigation of structural changes of β-casein and lysozyme at the gas-liquid interface during foam fractionation

Purification and separation of proteins play a major role in biotechnology. Nowadays, alternatives to multistep operations suffering from low product yields and high costs are investigated closely amidst which one of the promising options is foam fractionation. The molecular behavior at the gas-liquid interface plays an important role in the formation and stabilization of enriched foam. This study for the first time correlates the physico-chemical parameters to the molecular structure in view of protein enrichment during foam fractionation of the two relatively different proteins lysozyme and β-casein employing biophysical techniques such as circular dichroism (CD) spectroscopy and infrared reflection absorption spectroscopy (IRRAS). In case of lysozyme, high enrichment was achieved at pH相似文献   

Modulation of cytochrome C coupling to anionic lipid monolayers by a change of the phase state: a combined neutron and infrared reflection study

The effect of monolayer domain formation on the electrostatic coupling of cytochrome c from the subphase to a monolayer at the air/water interface was studied using a combination of neutron reflection (NR) and infrared reflection absorption spectroscopy (IRRAS) techniques. The monolayers consisted of a binary mixture of the zwitterionic phosphatidylcholine and the anionic phosphatidylglycerol. For a monolayer of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylglycerol (DMPG, 30 mol%), which exhibits a non-ideal mixing of the two lipid components, we observed a significantly higher protein coupling to the liquid-condensed phase compared to the liquid-expanded state. In contrast, this higher protein binding was not observed when the two lipids had identical chain lengths (nearly ideal mixing). Similarly, for an equimolar mixture of DPPC and DMPG, we did not observe significant differences in the protein binding for the two phase states. The results strongly suggest that the domain formation in a condensed monolayer under non-ideal lipid mixing conditions is crucial for the cytochrome c binding strength. Furthermore, this study demonstrates the significant advantages of gathering information on protein-monolayer coupling by the combined use of a dedicated IRRAS set-up with the NR technique.     

High-resolution circular dichroism of N-acetyl-L-phenylalaninamide: solvent effects

Biological membranes consist mainly of lipids and proteins. At present, the structure of the lipid phase appears to be established, but hypotheses on the molecular organization of the protein are difficult to support. Thus the deformation behavior of whole human erythrocyte ghosts, ghosts after the selective removal of lipids and ghosts stripped of lipids as well as nonlipid components have been examined in the hope of securing indirect information on the organization of the protein. It has been found that large localized deformations result in partial membrane failure and long uniformly wide fibrils, frequently in excess of 3000 Å, are drawn across the rupture. These data are interpreted in terms of currently favored membrane models and the possibility of a fibrous membrane framework consisting predominantly of protein is reviewed. The behavior of the membrane in its various stages of extraction is compared and contrasted to that of synthetic polymer films of known organization.     

A thermodynamic and structural study of myelin basic protein in lipid membrane models

Myelin basic protein (MBP) is a major protein of the myelin membrane in the central nervous system. It is believed to play a relevant role in the structure and function of the myelin sheath and is a candidate autoantigen in demyelinating processes such as multiple sclerosis. MBP has many features typical of soluble proteins but is capable of strongly interacting with lipids, probably via a conformation change. Its structure in the lipid membrane as well as the details of its interaction with the lipid membrane are still to be resolved. In this article we study the interaction of MBP with Langmuir films of anionic and neutral phospholipids, used as experimental models of the lipid membrane. By analyzing the equilibrium surface pressure/area isotherms of these films, we measured the protein partition coefficient between the aqueous solution and the lipid membrane, the mixing ratio between protein and lipid, and the area of the protein molecules inserted in the lipid film. The penetration depth of MBP in the lipid monolayer was evaluated by x-ray reflectivity measurements. The mixing ratio and the MBP molecular area decrease as the surface pressure increases, and at high surface pressure the protein is preferentially located at the lipid/water interface for both anionic and neutral lipids. The morphology of MBP adsorbed on lipid films was studied by atomic force microscopy. MBP forms bean-like structures and induces a lateral compaction of the lipid surface. Scattered MBP particles have also been observed. These particles, which are 2.35-nm high, 4.7-nm wide, and 13.3-nm long, could be formed by protein-lipid complexes. On the basis of their size, they could also be either single MBP molecules or pairs of c-shaped interpenetrating molecules.