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* School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia; and Department of Materials, University of Oxford, Oxford OX1 3PH, United Kingdom
Correspondence: Address reprint requests to Dr. Ian Gentle, School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia. Tel.: +61-7-3365-4800; Fax: +61-7-3365-4299; E-mail: i.gentle{at}uq.edu.au.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND PROCEDURESRESULTS AND ANALYSISDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND PROCEDURESRESULTS AND ANALYSISDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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; Review Issue of Comparative Biochemistry and Physiology, 2001; Review Issue of Pediatric Pathology and Molecular Medicine, 2001). Components in this fluid are recognized as critical to various aspects of lung function, the most obvious of which is the need to preserve a very low surface tension at the air-fluid interface. A low surface tension prevents pulmonary collapse such as is associated with respiratory distress syndrome (RDS), and which is a common cause of death in premature infants. The primary surfactant associated with this low surface tension is the zwitterionic lipid dipalmitoylphosphatidylcholine (DPPC), though anionic lipids, in particular dipalmitoylphosphatidylglycerol (DPPG) are also known to have an important role (Oviedo et al., 2001; Takamoto et al., 2001). By themselves, these lipids do not have the transport properties required in order to accommodate changes in the area of the alveolus during the breathing cycle, and three of the four surfactant-associated proteins (SP-A, SP-B, and SP-C) have been implicated as having roles in these processes (Casals, 2001; Haagsman and Diemel, 2001).
The most enigmatic of the surfactant proteins is SP-B, which forms the subject of this work. It is the only surfactant protein critical to lung function, as shown by animal studies, and is a disulfide-linked homodimer, each half consisting of 79 amino acids. An excellent review of its known properties is given by Weaver (Weaver and Conkright, 2001). It has a strong overall positive charge at physiological pH. A structural model has been presented by Zaltash which is based on similarities with NK-lysin (Zaltash et al., 2000), a simplified version of which was presented earlier by Cruz (Cruz et al., 1995). Essentially, in the model of Zaltash, the protein consists of five amphipathic 
-helices, folded such that the protein's hydrophobic faces are buried. The relative positioning of the helices is largely constrained by three internal disulfide bonds within each monomer. Given its critical role in normal lung function, and highly conserved primary structure, it is probable that SP-B's modus operandi does not vary from one species to the next. In what follows we provide evidence to suggest that SP-B unfolds to present its hydrophobic interior when present as a monolayer at the interface, and propose that this conformational change plays an important part of its normal duty cycle in the presence of lipids.
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MATERIALS AND PROCEDURES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND PROCEDURESRESULTS AND ANALYSISDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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) of the method of Curstedt (Curstedt et al., 1987). Fluid from lavaged or minced lungs was centrifuged at 20,000 x g for 30 min before extracting the pellet into chloroform/methanol according to the procedure originally described by Bligh and Dyer (Bligh and Dyer, 1959). The lower (organic) phase was evaporated to a few milliliters, applied to an 80 x 2 cm column of Sephadex LH-20 and eluted using a 2:1 CHCl3:methanol (v/v) solvent mixture while monitoring the absorbance at 280 nm. The first peak from this column contained the hydrophobic proteins, which were pooled, dried under N2, and immediately redissolved in 
5 mL of 1:1 CHCl3:methanol + 5% 0.1 M HCl(aq). This was applied to a 60 x 2 cm Sephadex LH-60 size-exclusion chromatography column and eluted using the latter solvent system under 2 m head of pressure. This second column effected a separation of SP-B, SP-C, and remaining lipid components comparable to that observed by Pérez-Gil (Pérez-Gil et al., 1993). Analysis of the putative SP-B fractions by SDS-PAGE (14% tris-tricine) stained using Coomassie blue showed a major protein band corresponding to an apparent molecular mass in the range of 20–30 kDa. Under reducing conditions (ß-mercaptoethanol) the apparent molecular mass reduced to 5–6 kDa. Peptide sequencing was performed on the Western blot transfer of one such gel, which confirmed that SP-B was present, in addition to contaminants including - and ß-hemoglobin. Electrospray mass spectral analysis indicated that sheep and cow SP-B exist as dimers with molecular masses of 17,430 Da (sheep) and 17,450 Da (cow). The latter result is consistent with that of Nag (Nag et al., 1999).
Bovine SP-B was used in neutron reflectivity measurements, whereas for the x-ray reflectivity measurements SP-B from both sheep and cows was used. The subphase used was either 0.15 M NaCl containing 0.01 M HEPES (unadjusted pH 5.5), or 0.10 M NaCl containing 0.01 M HEPES, (pH 7.2). Neutron scattering gives the opportunity to vary the subphase contrast by partial or complete deuteration of the water, and subphases of D2O, 50% H2O:D2O mixtures (elsewhere referred to as HDO), and air contrast-matched water (elsewhere referred to as ACMW, composition H1.842D0.158O) were used. Calculation of titration curves indicated that for a pH change from 5.5 to 7.2 the charge of the dimeric protein would change from +11.5 to +10.1 in either species. (Database and facilities are available via http://au.expasy.org/.) It therefore seems unlikely that the modest differences in salinity or pH would lead to significant differences in the interfacial behavior of SP-B. Films were spread directly from LH-60 eluent solutions, and all measurements were done at 22.5°C.
Reflectometry measurements were carried out on Teflon Langmuir troughs (NIMA Technologies, Coventry, UK) using the angle-dispersive x-ray reflectometer (Schlossman et al., 1997) at beamline X-19C of the National Synchrotron Light Source, NY, USA, (
= 1.54 Å), and neutron measurements were performed on the energy-dispersive CRISP reflectometer (Penfold et al., 1987) at the ISIS spallation source at Rutherford Appleton Laboratory, Didcot, UK (angle of incidence = 1.5°). Surface pressure-area measurements complement each reflectivity profile. Because the angle-dispersive x-ray reflectivity curves cover many orders of magnitude in intensity, and to compensate for changes in the x-ray beam footprint at the interface as a function of angle, these data were collected in (typically) nine regions of momentum transfer Q = (4
/)sin
. An appropriate combination of input slit width, measurement time, and brass attenuators in front of the detector was used for each region. These data regions were made to overlap slightly in order to permit their subsequent rescaling. Analysis of the reflectivity curves was performed using the programs Parratt32 (Hahn Meitner Institute), SURFace (see http://www.isis.rl.ac.uk/largescale/SURF/technical/surface.htm) or Wincxmulf (Wincxmulf is a version of software developed at ISIS and adapted for multiple constrained refinements (Wincxmulf_1) by A.S. Brown (Research School of Chemistry, Australian National University)), which are based on optical matrix formalisms (Brown et al., 1997; Penfold and Thomas, 1990). Although each has particular strengths and weaknesses, results are invariably comparable, so that the choice of program is essentially dictated by convenience.
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RESULTS AND ANALYSIS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND PROCEDURESRESULTS AND ANALYSISDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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; Krol et al., 2000; Oosterlaken-Dijksterhuis et al., 1991; Taneva and Keough, 1994; Taneva et al., 1998), and when plotted on the same scale they show a very considerable variation in limiting area (from 20–120 nm2 per dimer). Large variations exist even for material originating from the same species. This almost certainly arises from difficulties in establishing the concentration of the spread chloroform/methanol SP-B solutions. Taneva and Keough have carefully addressed the difficulty of obtaining accurate protein concentrations and therefore limiting areas in studies of porcine SP-B (Taneva and Keough, 1994). Since SP-B from different species has very similar primary structure (and therefore presumably higher structure also), we have opted to treat the concentration of our protein solutions as a variable parameter, which can be adjusted so that our SP-B isotherms agree with the isotherm published by Taneva under almost identical conditions. Examples of sheep and cow SP-B isotherms that have been fitted in this way are shown in Fig. 1.
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30–40 mN m-1. This plateau, which occurs at 15 nm2 molecule-1 in the illustrated isotherms, coincides with the first appearance during compression of faint visible streaks in the monolayer parallel to the advancing barrier and within a few centimeters of it. This observation demonstrates that the plateau corresponds to collapse of the protein monolayer which subsequently remains associated with the surface, an argument supported by the relatively small loss of material on subsequent slow cycles in Fig. 1. It also argues against attempting to interpret reflectometry results at smaller areas, as the surface will contain features on a length scale at least comparable to the wavelength of visible light. At such small areas the film loses its fluidity, so that the Wilhelmy plate is pushed in the direction of the barrier movement and will give a false reading. This is the likely explanation for apparent changes in the surface pressure at the smallest areas shown in Fig. 1. Further experiments (data not shown) demonstrate that the collapse pressure is independent of temperature over the range 15°C–45°C.
X-ray reflectivity
An example of an x-ray reflectivity profile and the corresponding fitted scattering-length density (SLD) profile is shown in Fig. 2. The x-ray models were generated by the following procedure, which we refer to as the discrete density profile (DDP) method. The procedure is equally applicable to neutron data, however these were modeled using the conventional box-models as described later. A model is established consisting of zero, one, two, or more layers, each with the same thickness t, and with roughness parameters 
constant throughout. We fix 
1/Qmax, and t 3, where Qmax is the maximum momentum transfer at which coherent scattering is observable in the data set in question. For neutrons this would be the point at which the observed reflectivity has dropped to the level of the incoherent background scatter, whereas for x-rays it is rather more subjective, with error bars typically obscuring details for Q > 0.6 Å-1. The SLD of each layer is initially set to that of the subphase (ie. a reasonable starting estimate which introduces no steps except at the air interface where the most distinct step is anticipated) and then only the SLDs of the layers are refined. In this way the best model is the one that satisfactorily models the data using the least number of layers and is amenable to chemical interpretation. In practice we find it is almost invariably possible to quickly produce an excellent and chemically meaningful fit to our reflectivity data in this way; in related work we find that it readily reproduces the anticipated SLD profiles for phospholipids at interfaces, which are more commonly found by two-layer models in which thicknesses, roughnesses, and SLDs are all refined.
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/Qmax, where dmin is the minimum distance over which the data can resolve SLD gradients. These gradients are characterized in most reflectivity fitting packages by convolution of step functions with Gaussians of standard deviation (the roughness parameter). By fixing all such roughnesses in a model such that 3 dmin, we ensure that we will not refine profile features sharper than the available data can resolve. Given that 3 dmin /Qmax, the assignment 1/Qmax is reasonable. Consider digitizing an arbitrary SLD profile into a number of slabs of thickness t, each with edges characterized by the parameter . In this way, a large number of very thin slabs would lead to a large number of parameters needing simultaneous refinement; moreover if the slabs were very thin they would be entirely smeared out after convolution with the Gaussian. On the other hand, very thick slabs will not permit digitization of an arbitrary SLD profile. The choice t 3 represents a suitable compromise. The approach proves very useful for systems involving monolayers whose total anticipated thickness is only several times greater than the calculated value of t. For thicker systems the number of layer SLDs requiring simultaneous refinement becomes unmanageable, with a corresponding growth in the number of local minima. In this situation one can do preliminary refinements using suitably thick and smeared layers to reduce the number of initially fitted densities and obtain a gross impression of the structure, then subdivide them and reduce the roughnesses until the instrumental resolution is obtained. This approach was not necessary in the profile models presented here, but has been shown to work nevertheless. Such models sometimes find their way into a local minimum; we find that this can often be addressed by temporarily changing the weighting scheme (Parratt32) or fitting algorithm (Wincxmulf) used to fit the data, though this was not necessary in any of the fits presented here. Fits presented here always minimize residuals calculated using the error bars obtained in the experiment.
The SLD profile found in this way can often be satisfactorily represented using a smaller number of layers of fitted thickness, SLD, and roughness in a way that requires fewer parameters and agrees with chemical intuition. In this context, situations commonly arise in which something is known about the system that can help pin down one or more parameters. For example, monolayer roughnesses at the air interface are unlikely to greatly exceed 3 Å in phospholipid systems at high pressure, and synchrotron x-ray data confirms this (Helm et al., 1991). In fitting phospholipid neutron reflectivity data, in which the resolution is much poorer, the use of such a low value is otherwise very difficult to justify. Although permissible under such circumstances, we have not yet encountered a reflectivity profile in which it is impossible to produce a comparably good fit by direct application of the DDP method. Moreover, there are situations in which a symmetrically rough edge as refined by traditional layer approaches is inappropriate. The air-protein and protein-subphase interfaces observed in the x-ray SLD profiles in this work are examples of this, in which the DDP method permits a considerably more flexible and presumably realistic assessment of the form of the electron density gradient.
The fitted x-ray SLD profiles of ovine and bovine SP-B monolayers are generally comparable at similar surface pressures (see Fig. 4), consisting of a density maximum that broadens and moves to a greater depth as collapse of the film is approached. This strengthens our belief that bovine SP-B, though possibly structurally different from sheep and other sequenced mammals, essentially operates by the same mechanism.
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). In many individual cases satisfactory fits can be obtained by simply fitting an air-water roughness, or by allowing the air SLD to decay exponentially to the subphase SLD (available in Parratt32) with the only fitted parameter being the decay length. Synchrotron x-ray data, with its relatively small error bars and extended Q-range, necessitates much more critical fitting. Unfortunately the DDP method cannot be fruitfully used in simultaneous fits of the x-ray and neutron data because the thicknesses are not fitted. Turning to more traditional box methods, it must be remembered that the protein's x-ray SLD profile (which will have a significant contribution from the protein's sulfur atoms) is in all likelihood rather different from its neutron SLD profile (for which we anticipate a more uniform distribution, with a higher potential for proton/deuteron exchange in the hydrophilic parts of the molecule), so that without a strong impression of the structure and orientation of the protein, it is unclear how to mutually constrain the resulting layers. Simultaneous reflectivity fits based on evolution of molecular models (Politsch, 2001) are probably the long-term answer to this dilemma.
In the meantime, an appropriate compromise is to model the x-ray and neutron data independently. X-ray data was fitted using the DDP method, then models of the neutron data were fitted in which layer thicknesses were mutually constrained, with edge roughnesses fixed at the smallest value sensibly inferred from x-ray fits. Fig. 3 shows an example of such a neutron fit, in which the neutron roughnesses have been fixed at 3.0 Å (a value comparable to capillary waves in such systems (Braslau et al., 1988; Daillant et al., 1990; Schalke et al., 2000; Schalke and Losche, 2000)). In Fig. 4 the SLD profiles obtained in this way are compiled to permit meaningful comparison, whereas Table 1 lists the corresponding fitted neutron parameters.
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260 per bovine dimer); it can be assumed that the mole fraction of D2O in the subphase (
D) determines the extent of this exchange, which is essentially at equilibrium within 15 min (Pastrana-Rios et al., 1995). We can estimate the number density of protein molecules at the interface using the molecular area provided by isotherms (Am) and the fitted layer thickness (tf) provided by the simultaneously fitted neutron models (for simplicity we only consider the layer at the air interface). This leaves us in a position to evaluate every term in the following expression for the scattering-length density (S) except nw, the number of molecules of included water per protein molecule:
 | (1) |
 | (2) |
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prot. This can be conveniently estimated by analogy with a chemically similar substance, such as the polyamide nylon, density 1.2 g cm-3. Molecular volume arguments permit the following expression for Fw:
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 | (5) |
Table 2 shows a clear reduction in the amount of included water as the film is compressed, as one might expect. On the other hand, the fractional volume occupied by the water appears rather high for such a hydrophobic protein, suggesting that it does not pack neatly at the interface. This is likely due in large measure to mutual repulsion caused by the strong positive charge of the protein (+10 per dimer at pH 7), a notion supported by the work of Holt (Holt et al., 2000), Su (Su et al., 1998, 1999) and Lu (Lu et al., 1999) who investigated the pH dependence of the density.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND PROCEDURESRESULTS AND ANALYSISDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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) in 1987. Based on that sequence, it has been proposed (Haagsman and Diemel, 2001; Johansson et al., 1991) that the structure of SP-B might be unusual in cows, owing to the relatively small proportion of cysteine in comparison with SP-B from other species. We note that the original assignment of several bovine residues was tentative, in particular residues 46 (an otherwise conserved cysteine linked to Cys-35) and 48 (the cysteine invoked in homodimer formation). It has been reported that the Cys-35/Cys-46 disulfide bond is critical for intracellular trafficking of SP-B (Beck et al., 2000), whereas our own and other (Nag et al., 1999) mass spectral results indicate that bovine SP-B is dimeric through disulfide bonds (Yu et al., 1987). The similarity of our results for bovine and ovine SP-B is consistent with the proposition that the protein sequences and structures are largely conserved.
Structural model
To date there is little direct evidence for the tertiary or quaternary structure of native SP-B, but the available data points to the structure described by Zaltash (Zaltash et al., 2000). This model is based on structural similarities to NK-lysin (Andersson et al., 1995), a protein for which a solution-phase NMR structure has been obtained (Liepinsh et al., 1997). The N- and C-terminal helices (helices 1 and 5, respectively) in each monomer are locked in an antiparallel arrangement by a pair of disulfide bonds, whereas helices 2 and 3 are kept in an antiparallel hairpin arrangement by another internal disulfide bond. Interestingly, the tertiary structure proposed by Zaltash does not present an obviously hydrophobic face to its external environment; because the five helices wrap onto each other in such a way that the hydrophobic stretches on the amphipathic helices are all in mutual contact.
Physical models suggest that this tertiary structure can be unfolded into a flat structure which for each monomeric half looks approximately like a boomerang, with helices 1 and 2 (at the N-terminus) forming the concave edges and helices 3, 4, and 5 forming the convex edges (see Fig. 5). One face of this unfolded structure is almost entirely hydrophobic, whereas the other face is largely hydrophilic, and it is clear how such an amphiphilic structure would orient at an air-water interface. Such unfolding would approximately double the area presented by the protein at the air-water interface.
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) in such a way that the protein is unable to present the hydrophilic faces of both monomeric halves to the subphase at the same time. It is possible that the acid-base pair breaks when the protein is spread at the interface in order to enable this. There are only two acidic residues in the largely basic SP-B, and both are conserved, though the location of one of them varies from residue 59–63. Perhaps the latter acidic residue has a role to play in the flattened version of SP-B, or in interactions with the zwitterionic DPPC.
Isotherms
There have been relatively few studies of native SP-B at the air-water interface. A strong indication that the -helices lie flat at the interface at low pressure comes from the limiting areas of Dieudonne's isotherms of peptides SP-B1–20 and SP-B9–36, both of which involve amphipathic -helices (Dieudonne et al., 2001). Extrapolating those results (0.21 nm2 residue-1) to the 158 residues in the native dimeric protein suggests that it would occupy 33 nm2 per dimer if the helices were all laid flat at the interface as in Fig. 5. For their globular NK-lysin-based SP-B dimer model, Zaltash (Zaltash et al., 2000) describes "approximate (linear) dimensions of 55 x 37 Å," pointing to a close-packed molecular area in the vicinity of 15 nm2 per dimer. We therefore propose that the 15
40 nm2 dimer-1 region of the isotherms in Fig. 1 (in which our protein concentration was scaled to match Taneva's (Taneva and Keough, 1994) porcine SP-B isotherm) represents the reversible pressure-induced folding of the protein.
In the 1540 nm2 dimer-1 region the SP-B isotherms are linear with a shallow slope (0.5 mN m-1 per 1% change in molecular area). By way of a familiar comparison, DPPC isotherms are very steep in the condensed-phase region as a result of close packing of the hydrocarbon chains (2.6 mN m-1 per 1% change in molecular area, data not shown). The comparison suggests that the folding of SP-B into its high-pressure conformation is a gradual process as the pressure increases. The integral under the isotherm represents the system's energy change in going from molecules spread out at the interface to balled-up molecules. From the baseline to the point of collapse the isotherm is fairly linear, allowing us to estimate this integral as 4 x 10-19 J molecule-1 or 260 kJ mol-1.
There is considerable hysteresis associated with the collapse at 36 mN m-1 from which it is clear that the moderate rate of compression used (20 cm2 min-1 on a 300 cm2 trough) was rather too fast for the kinetics of the system. That this hysteresis is largely due to structural changes associated with the collapse is clear from the relative lack of hysteresis in the final short cycle presented in Fig. 1, in which the collapse area was not attained.
Support for this model from x-ray reflectivity
The fitted SLD profiles in Fig. 4 show fairly clearly that at surface pressures below 20 mN m-1 the most electron-dense part of the protein monolayer is within 10 Å of the air interface. Such SLD profiles are remarkably similar to those observed by Holt (Holt et al., 2000) for myoglobin at interfaces, who proposed changes in the tertiary structure of that protein such that its amphipathic -helices were laid flat. In our model illustrated in Fig. 5, this region will correspond to the location of electron-dense regions of the protein monolayer, in particular the protein's relatively electron-rich disulfide linkages.
At pressures above 20 mN m-1 but below the collapse at 36 mN m-1, the SLD profiles show a much shallower electron density gradient from the air toward the maximum, which now lies 20 Å from the air interface. At such pressures we believe that the protein is essentially in the globular conformation proposed by Zaltash, with both halves of the dimer at the interface. There is an orientation of the dumbbell-shaped dimer which would position almost all the sulfur in the protein at such a considerable depth from the surface; this has the dimer's C2 axis coincident with the surface normal, with helix 3 of each monomer almost parallel to it. It is harder to determine whether the N-termini of helices 3 would be up or down, since the sulfur atoms are located fairly centrally within the folded protein. However, we tentatively propose that the N-termini of helices 3 are uppermost on the grounds that it is easier to see how the protein would end up in this orientation at the conclusion of the process of folding from the flat structure (Fig. 5). One can be confident that recent advances in reflectivity fitting based on molecular modeling (Politsch, 2001) will greatly assist such interpretations in the future.
One must be extremely wary of over interpreting the SLD profile in cases where the contrast with the subphase becomes very poor. Unfortunately, this renders the true thickness of the protein layer somewhat speculative because the hydrophilic parts of the protein, which can be expected to be at the interface of the protein with the subphase, will contain an increasing proportion of included solvent as well as a relatively high proportion of exchangeable protons. Consequently we do not expect the boundary to be distinct from the standpoint of reflectivity.
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CONCLUSIONS |
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), and we are able to tentatively assign an orientation for the protein in this state. At molecular areas less than 15 nm2 molecule-1, the monolayer visibly collapses, but remains associated with the interface. The neutron-reflectivity results indicate the exchange of the protein's labile protons as well as a very substantial amount of included water in the protein monolayers.
We draw attention to the fact that the model proposed here takes no account of the protein's interaction with lipids (a topic of primary importance in natural lung surfactant which will be addressed in a subsequent publication). Instead, it serves to suggest the conformational flexibility of the protein in a way that we trust will lead to models for its interaction with lipids. We propose that such conformational changes are crucial to the functionality of SP-B since they point to a mechanism by which cycling of other surfactant components could occur, involving the reversible pressure-induced interment of amphipathic helices in the subphase. This work will also be of interest in the study of other amphipathic proteins (Weinberg et al., 2000).
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ACKNOWLEDGEMENTS |
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) available to us. This work was enabled by an Australian Synchrotron Research Program Fellowship awarded to W.K.F, and access to synchrotron and neutron scattering facilities was possible thanks to funding provided by the Australian Access to Major Facilities program.
Submitted on January 13, 2003; accepted for publication June 17, 2003.
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, 
, respectively. The entire sequence required a little over an hour. The relative lack of hysteresis in the third cycle (•, 
), which stops shy of the collapse at 
), the barrier was stopped for a few minutes at 
