Exclusion of SP-C, but not SP-B, by gel phase palmitoyl lipids

Chemistry and Physics of ELSEVIER

Chemistry and Physics of Lipids 76 (1995) 27-39

LIPID$

Exclusion of SP-C, but not SP-B, by gel phase palmitoyl lipids Ann D. Horowitz* Division of Pulmonary Biology, TCHRF 4024, Children's Hospital Medical Center, 3333 Burner Ave., Cincinnati, Ohio 45229-3039, USA

Received 20 October 1994; revision received 17 January 1995; accepted 6 February 1995

Abstract The interactions of the hydrophobic pulmonary surfactant proteins, SP-C and SP-B, with lipid bilayers were assessed by fluorescence energy transfer. SP-C and SP-B were labeled with the fluorescent probe, succinimidyl nitrobenzoxadiazolyl amino hexanoate (NBD). Fluorescence energy transfer from NBD-SP-C and NBD-SP-B to four distinct indocarbocyanine probes (C, DiI) was utilized to determine the association of the surfactant proteins with various lipid acyl chains. In lipid mixtures including DPPC and DPPG, SP-C was associated with shorter chain and unsaturated lipids below the bulk lipid phase transition. Longer chain saturated CnDiI were excluded from SP-C aggregates. In contrast, SP-B demonstrated little acyl chain preference. The association of SP-C with shorter chain and unsaturated lipids below the bulk phase transition is interpreted to arise from a mismatch in the length of the hydrophobic region of the SP-C ~-he.lix relative to the length of the hydrophobic region of dipalmitoyl lipids in the gel phase. Keywords: Pulmonary surfactant; Acyl chain length; Energy transfer; Indocarbocyanine dye

1. Introduction Pulmonary surfactant is a lipid-protein complex which lines the alveolar surface in the lungs, lowering surface tension and preventing collapse of the distal components of the lung [1,2]. De-

creased pulmonary surfactant is associated with respiratory distress syndrome (RDS) in premature infants and is now routinely treated by surfactant replacement with preparations derived from organic extracts of animal surfactant. Phosphatidylcholine (82%) and phosphatidyl glycerol (5%),

Abbreviations: C/M, chloroform/methanol; Tin, melting temperature; PC, phosphatidyl choline; PG, phosphatidyl glycerol; DMPC, dimyristoyl-PC; DPPC, dipalmitoyl-PC; DPPG, dipalmitoyl-PG; POPC, l-palmitoyl,2-oleoyl-PC;POPG, l-palmitoyl,2oleoyl-PG; NBD, 6-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)aminohexanoyl;ET, fluorescence energy transfer; CnDiI, diacyl 3,3,3',3'tetramethylindocarbocyanine perchlorate, where n designates the number of carbons in the acyl chains; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; HEPES, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonicacid; SL lipids, 21.6:5.3:3.5:1:1:0.8/DPPC/POPC/DPPG/DMPC/DSPC/POPG (w/w). * Corresponding author, Tel.: (513)-559-8919.

0009-3084/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSD! 0009-3084(94)02426-J

28

A.D. Horowitz / Chemistry and Physics of Lipids 76 (1995) 27-39

and lesser amounts of other lipid classes [3] are the predominant components of pulmonary surfactant. Approximately 80% of the acyl chains are palmitoyl [2]. Organic extracts of surfactant also contain two small hydrophobic proteins, SP-B and SP-C, which greatly enhance the surface spreading and stability of the lipid components both in vivo and in vitro [4-7]. Surfactant-associated proteins SP-B and SP-C are highly hydrophobic surface-active molecules. Bovine SP-C contains 34 amino acid residues of which 23 are valine, leucine or isoleucine [4,8,9]. Two palmitic acid residues are attached via thioester linkages to two adjacent cysteines in the N-terminal region of SP-C [10]. Residues 12 to 32 contain exclusively nonpolar amino acids. Although a dimeric form of SP-C which consists predominantly of fl-sheet secondary structure has been described [11], SP-C exists primarily as a monomer, with a secondary structure containing a high proportion of or-helix. The proportion of SP-C in an or-helical conformation was determined by Fourier transform infrared spectroscopy (FTIR) to be between 46.5% and 90% [12-14], and between 46% and 86% by circular dichroism [15]. In oriented lipid bilayer films, the axis of the u-helix lies approximately parallel with the lipid acyl chains [13,14], implying a transbilayer orientation. The structure of porcine SP-C in chloroform/methanol (C/M) solution was determined by nuclear magnetic resonance [16]. The region from residue 8 through 35 forms a very regular u-helix. The length along the helix axis of the exclusively aliphatic region, residues 13-28, is 23/~ [161. SP-B contains two positively charged amphipathic helices [8], which are crosslinked by three intramolecular disulfide bridges [17]. An additional cysteine is free to form intermolecular disulfide bonds. The amphipathic nature of SP-B's u-helices suggest that SP-B rests at the surface of the lipid bilayer. SP-B and SP-C enhance the surface active properties of lipid mixtures by lowering surface tension and enhancing adsorption rate of a lipid film at the air/water interface [7,18-20]. When present in a pre-formed lipid monolayer, SP-C and SP-B increase the rate of phospholipid inser-

tion from lipid vesicles injected into the subphase [21]. SP-C lowers the phase transition temperature, Tm, of DMPC and DPPC/DPPG lipid vesicles and broadens the phase transition [22,23], as assessed by anisotropy of fluorescent lipid probes. However, differential scanning calorimetry results concerning the effects of SP-C on lipid bilayer vesicles are contradictory, SP-C was reported to lower the Tm of DMPC vesicles [24], but to raise the Tm of DPPC and DPPG vesicles [15]. The effect of SP-B on the lipid structure of DPPC/ DPPG vesicles was studied by a variety of fluorescent probes [25]. SP-B increased the Tm of DPPC/DPPG vesicles, as observed by anisotropy of diphenylhexatriene or cis-parinaric acid, however, trans-parinaric acid, which partitions preferentially into the gel phase, detected no change in Tm arising from incorporation of SP-B. The aggregation of SP-C in the presence of gel phase lipids was demonstrated using fluorescence energy transfer (ET) [12]. The lowering and broadening of the phase transition by SP-C suggests that the lipids surrounding SP-C within these aggregates may be in a more 'fluid-like' state than the bulk lipid [22]. Studies of model peptides which contain hydrophobic c~-helical transmembrane domains have shown that segregation into peptide-rich and peptide-poor regions occurs when the hydrophobic region of the peptide is much shorter than the lipid hydrophobic region [26,27]. Aggregation occurs more readily in gel phase lipids because the rigidity of the bilayer in the gel phase accommodates the hydrophobic mismatch poorly [28]. In the present experiments, the association of SP-C with lipids containing various acyl chains was tested using indocarbocyanine dyes differing only in their acyl chains. Indocarbocyanine dyes (C, DiI) are selective for fluid or gel phase lipid domains, depending on the hydrocarbon chain length of the indocarbocyanine dye relative to that of the bulk lipid [29,30]. At high CnDiI dye concentrations (10 %M), CnDiI of a chain length much shorter than that of bulk lipids form self-quenched domains below the phase transition temperature of the lipid, resulting in a large decrease in fluorescence. CnDiI dyes of chain lengths the same or slightly greater than that of the bulk lipids do not show this behavior;

A.D. Horowitz / Chemistry and Physics of Lipids 76 (1995) 27-39

their fluorescence increases at the transition of the bulk lipid to the gel phase [29]. The gel/fluid partition coeflicients of various C, DiI determined using fluorescence quenching by a spin-labeled lipid [30], confirming that the C, DiI partition into coexisting lipid phases according to the length of their acyl chain:~ relative to the lipid acyl chain length. Thus the, C, DiI comprise a set of probes which differ only in their preference for the fluid or gel phase, in a predictable manner. In order to determine the nature of the lipids immediately surrounding SP-C and SP-B, we have used ET between fluorescently labeled SP-C or SP-B and C12Di][, C16DiI, ClsDiI and A9A2ClsDiI. SP-C and SP-B were labeled with the fluorescent probe, succinimidyl NBD hexanoate. The degree of association of NBD-labeled SP-C (NBD-SP-C) or NBD-labeled SP-B (NBD-SP-B) molecules with C, DiI in multilamellar lipid vesicles was monitored by quenching of donor (NBD) fluorescence in the presence of aceeptor (C~DiI). Multilamellar vesicles were used because they more closely resemble the multilamellar structures present in native surfactant, and in lamellar bodies of pulmonar.¢ Type II epithelial cells.

2. Experimental procedures 2. I. Materials Methanol and chloroform were optima grade from Fisher Scientific (Cincinnati, OH). All lipids were from Avanti Polar Lipids (Alabaster, AL), except for DMPC and DSPC, which were from Sigma Chemicall Co. (St. Louis, MO). Succinimidyl NBD hexanoate, and indocarbocyanine dyes (C, DiI) were from Molecular Probes Inc. (Eugene, OR). Column chromatography material was Bio-Sil HA minus 325 mesh from Bio-Rad Co. (Richmond, CA), lipophilic Sephadex LH-60, and reverse phase C-8 silica from Sigma Chemical Co. 2.2. Purification of SP-C and SP-B SP-C was isolated from cow lungs by methods described previously [12]. Briefly, freshly excised lungs were lavaged with 0.15 N saline and surfactant was pelleted by centrifugation. Surfactant was extracted with C/M, and the extract was

29

dialyzed against the same solvent. SP-C and SP-B were purified from the dialysate by passage through a silicic acid column using a step-wise gradient of C/M. Alternatively, passage through a liquid chromatography column containing C-8 silica in 1:1 C/M (v/v), 1 mM HCI was utilized as a first chromatographic step. The fractions containing SP-C and SP-B were identified by SDSPAGE. The individual proteins were further purified by chromatography on lipophilic Sephadex LH-60 in 1:1 C/M (v/v) containing 1 mM HCI. Prolonged dialysis against the latter solvent was utilized to remove contaminating lipids. Column chromatography and dialysis were repeated until sufficiently pure preparations of SP-C and SP-B were obtained. In the final dialysis step, the purified SP-C was dialyzed against 2:1 C/M (v/v) to remove the HCI. SP-C was stored as a C/M (2:1 v/v) solution. SP-B was dialyzed against absolute ethanol acidified with 50/d/1 of HC1 for storage. Purity of SP-C and SP-B was determined by SDS-PAGE [31] on 17% acrylamide gels and SP-C concentration was determined by amino acid compositional analysis [32]. SP-C generated by this methodology contained no detectable SPB as assessed by Western blot analysis using SP-B specific antisera. Purity of the SP-B preparation was determined by amino-terminal sequencing on a Porton gas phase sequencer.

2.3. Fluorescent labeling of SP-B and SP-C Methods of labeling and the characterization of labeled SP-C have been previously described [12]. Briefly, SP-C in 2:1 C/M (v/v) was mixed with a four-fold molar excess of NBD-succinimide in 2:1 C/M (v/v), which had been adjusted to pH 7.6 with 1.0 M HEPES. The reaction was allowed to proceed with stirring at room temperature overnight. A 25 gl aliquot of 1M Tris-HC1 was added to deplete excess reactant, and the mixture was dialyzed against 1:1 C/M (v/v). Labeled SP-C was separated from free NBD by chromatography on lipophilic Sephadex LH-60 in 1:1 C/M containing 0.01 M HCI, followed by dialysis against 1:1 C/M. Fractions containing NBD-labeled SP-C were identified by gel electrophoresis [31], followed by examination under ultraviolet illumination. SP-B was labeled with NBD-succinimide by

30

A.D. Horowitz / Chemistry and Physics of Lipids 76 (1995) 27-39

the same procedure as SP-C. Labeling efficiency was calculated using an extinction coefficient of 1.7 x 103 at 466 nm for NBD succinimide. Protein concentrations were determined by amino acid composition. Labeling ratios obtained were 1.1 dye/protein molecule for NBD-SP-C and 1.4 for NBD-SP-B. 2.4. Preparation of multilamellar vesicles Stock solutions of phospholipids in chloroform and CnDiI in ethanol were mixed with surfactant proteins in 2:1 C/M (v/v) solution to give the protein/lipid ratio desired. For ET experiments, the CnDiI/lipid ratio was 1:2000 (mol/mol), unless otherwise noted. For C, DiI self-quenching experiments, the C, DiI/lipid ratio was 1:10 (mol/mol). The protein/lipid mixtures were incubated at 50°C while being dried under a gentle stream of nitrogen followed by removal of residual solvent under vacuum. A 2-ml aliquot of buffer, consisting of 10 m M 3-(N-morpholino)propane sulfonic acid, 120 mM NaCI, 2 m M EDTA, pH 7.0 (MNE buffer) at 50°C, was added to the dried film followed by incubation for 30 min at 50°C. Multilamellar vesicles were formed upon vortexing for 30 s as described previously [22], and sonicated for 30 s in an EM/C bath ultrasonicator at room temperature in order to reduce aggregation of vesicles. In experiments utilizing A9,12C18DiI, the buffer was flushed with N2 prior to addition to the lipid film, and samples were maintained under N2 throughout the experiment. Column chromatography of lipid vesicles containing NBD-SP-C or NBD-SP-B was conducted on a 1.5 × 15 cm Sepharose 6B column in M N E buffer. Elution of fluorescently labeled protein was monitored at 520 nm, with excitation at 460 nm. Elution of vesicles was monitored by light scattering at 360 nm. Both NBD-SP-C and NBDSP-B eluted in the void volume of the column with the vesicles, with no detectable fluorescence eluting in the included volume. In order to determine whether hydrolysis of lipids occurred during drying of lipids and reconstitution of vesicles; lipids were extracted [33] from vesicles formed by the above procedure and analyzed by thin-layer chromatography. In addition, lipids (7:1 w/w D P P C / D P P G ) dried together with proteins at

55°C under N z were analyzed by thin-layer chromatography. No evidence of lipid hydrolysis was detected in either case. 2. 5. Energy transfer and fluorescence measurements Fluorescence measurements were conducted using a Perkin-Elmer LS-50 fluorimeter. The temperature in the sample chamber was controlled (_+ 0.5°C) using a Lauda circulating water bath. The samples were allowed to equilibrate at the initial temperature for at least 30 min, with gentle stirring. The temperature was decreased gradually over the course of the experiment beginning at 50°C. Energy transfer experiments were conducted as previously described [12]. Briefly, the fluorescence of four samples was compared: B, a background sample in which none of the components were fluorescent; D, a sample containing donor-labeled surfactant protein; A, a sample containing acceptor (CnDiI); and DA, a sample containing both donor and acceptor. In all sanlples the total surfactant protein concentration was maintained constant by inclusion of the appropriate amount of unlabeled SP-C or SP-B. Excitation of NBD was at 460 nm, with fluorescence measured at three wavelengths at which the contribution of the acceptor was minimal, 510, 515 and 520 nm. At the end of the experiment, vesicles were disrupted by addition of 10% Triton X-100 to a final concentration of 1%, with the temperature raised to 50°C to abolish energy transfer. Energy Transfer (ET) was calculated from the raw data as follows: first, the contribution of the background light-scattering was subtracted from all data. Differences in donor concentration between the D and DA samples were corrected using the donor fluorescence observed in 1% Triton X-100 at 50°C [34]. The ET efficiency E was then calculated at each wavelength according to: E = 1 - (FDA

-

-

FA)/FD

where Fo, FDA and F A are the fluorescence of the D, DA and A samples. ET calculated at the three wavelengths was averaged. Results of all fluorescence experiments are presented as the average of two experiments with deviations indicated by error bars.

31

A.D. Horowitz / Chemistry and Physics of Lipids 76 (1995) 27-39

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Fig. 1. Self-quenchingof C, DiI in SL lipid vesicles. Self-quenchingis plotted as fluorescence(F) of the sample divided by the fluorescence of the sample in 1% Triton X-100 at 50°C. The concentrations of the C, DiI were hl0 C, DiI/lipid (mol/mol). SP-C concentrations were 2% (by weight) or 1:250 protein/lipid (mol/mol). The temperature was gradually decreased from 48-50°C to 20°C over the course of the experiment. Panel A: O, C12Dil; ~7, C12DiIplus SP-C. Panel B: II, CI6DiI; A, C16DiI plus SP-C. Panel C: V, A9'I2ClaDil; [~, A9't2C18DiI plus SP-C. Panel D: A, CtsDiI; <5 CIsDiI plus SP-C. Corrected emission spectra for quantum yield and spectral overlap calculations were obtained as described [35]. Quantum yields of NBD-SP-C and NBD-SP-B in 1% Triton X-100 were obtained relative to Na fl~torescein in 0.1 N NaOH, using a quantum yield of 0.92 for fluorescein [36]. Quantum yields in lipid vesicles at 20°C and 50°C were calculated relative to the quantum yield in 1% Triton X-100. Ro, the distance at which ET between two isolated fluorophores is 50%, was calculated from the spectral characteristics of each donor/acceptor pair, using the donor quantum yields obtained ~s described above [12,37]. 3. R e s u l t s

Fluid phase or gel phase preferences of the C, DiI probes in DPPC or D M P C can be determined from their tendency to form self-quenched

domains below the phase transition temperature of the bulk lipid, when the C, DiI are present at sufficiently high concentration (0.1 mol fraction) [29]. In the present work, the behavior of C, Dils in the presence and absence of 2% SP-C was determined in a lipid mixture (SL lipids) approximating the composition of pulmonary surfactant (Fig. 1), i.e. 21.6:5.3:3.5:1:1:0.87 DPPC/POPC/ D P P G / D M P C / D S P C / P O P G (w/w). The aggregation state of SP-C in SL lipids was previously characterized [12]. At 0.1 mol fraction of CL2DiI and A9,12CIsDiI , self-quenching was observed below 42°C, consistent with formation of selfquenched domains of C12DiI and Ag'12CI8DiI. In contrast, the fluorescence of CI6DiI and C18DiI increased, indicating that Cl6DiI and C18DiI remained dispersed in the gel phase lipid. SP-C (2%) did not significantly alter the temperature or breadth of the fluorescence changes. However, the

32

A.D. Horowitz / Chemistry and Physics of Lipids 76 (1995) 27-39

fluorescence intensity of the C, DiI probes, with the exception Of CxsDiI, was increased in the presence of 2% SP-C. Additionally, we examined the behavior of the C, DiI in 7:1 DPPC/DPPG vesicles. As expected, the fluorescence of C~zDiI and A9"~2CIsDiI was quenched below 42°C, whereas that of C16DiI and C,sDiI increased (data not shown). The behavior of the C, Dils in the presence of 2% (w/w) SP-C was similar to their behavior in lipid alone, except for alterations in overall fluorescence intensity, similar to that seen in SL lipids (Fig. 1). Fluorescence energy transfer from NBD-SP-C to the C, DiI in 7:1 DPPC/DPPG vesicles is shown as a function of temperature in Fig. 2. A major difference in behavior of the carbocyanine dyes was observed close to the phase transition temperature of the bulk lipids. A large increase in ET from NBD-SP-C to C12DiI and A9"12ClsDiI occurred just below the phase transition temperature. There was a decrease in ET from NBD-SP-C to C~6DiI and to C18DiI in the same temperature region. A maximum in the ET from NBD-SP-C to C12DiI and to A9'I2ClsDil occurred at 40°C. Below 42°C in 7:1 DPPC/DPPG, SP-C forms aggre0.4

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Fig. 2. Energy transfer from NBD-SP-C to C, DiI in 7:1 DPPC/DPPG vesicles. Fluorescence energy transfer from NBD-SP-C to: ~', A9't2CIsDiI; vq, Ct2DiI; T, Ct6DiI; II, C18DiI. The NBD-SP-C concentration was 0.5% protein/lipid (w/w), or 1:1t00 (mol/mol)and the C, DiI concentrationswere 1:2000 (mol/mol) relative to bulk lipid.

gates or patches enriched in SP-C [12]. The current results indicate that the shorter chain and unsaturated C, DiI are associated with SP-C aggregates or patches at temperatures just below the phase transition temperature of the bulk lipid. C16DiI and C,8DiI are excluded from the region surrounding SP-C at these temperatures. These results imply that the lipids surrounding SP-C are sufficiently disturbed near the phase transition to accommodate lipids with acyl chains which are unsaturated or significantly shorter than the bulk lipid, and to exclude longer chain saturated lipids. Below 34°C the ET from NBD-SP-C to all four of the C, DiI increase to similar values. In DPPC or DPPG vesicles, ET from NBD-SPC to Cº2DiI or CI6DiI was similar to that observed in 7:1 DPPC/DPPG (data not shown), indicating the lack of lipid headgroup specificity for the changes in ET close to the lipid phase transition temperature. ET from NBD-SP-C to C~2DiI and to C16DiI was observed in 7:1 DMPC/ D M P G vesicles (data not shown). A similar, though smaller, increase in ET to C,2DiI occurred at 23°C, near the phase transition of the bulk lipid, whereas ET to Cl6DiI decreased at the same temperature, indicating that the differential behavior of the C, Dils depends on the bulk lipid phase transition. If SP-C is excluded from gel phase lipids, ET from NBD-SP-C to ClzDiI and to A9A2ClsDiI should increase below the DPPC/DPPG transition temperature in a lipid mixture containing a mixture of acyl chains including palmitoyl lipids. At the same temperatures, ET from NBD-SP-C to C16OiI and to C18DiI should not increase. In the SL lipid mixture, SP-C aggregates below 37°C [12]. In this lipid mixture, a striking difference among the C, DiIs was apparent (Fig. 3). Below 37°C the ET vs. temperature curves for the carbocyanine dyes diverged. A large increase in ET from NBD-SP-C to C~2DiI and A9'I2CI8DiI was observed with decreasing temperature, while ET from NBD-SP-C to C16OiI and CIsDiI decreased. These results support the conclusion that SP-C is excluded by palmitoyl lipids in the gel phase, and is found in association with shorter chain and unsaturated lipids. The concentration of shorter chain lipids in the aggregates of SP-C could in

A.D. Horowitz / Chemistry and Physics of Lipids 76 (1995) 27-39

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Temperature (*C) Fig. 3. Energy transfer from NBD-SP-C to C, DiI in SL lipid vesicles. Fluorescenceenergy transfer from NBD-SP-C to: A, A9,12C18DiI; V], CtEDiI; T, Ci6DiI; II, C18DiI. The NBD-SPC concentration was 0.5% protein/lipid (w/w), or l:1100 (mol/ mol) and the C, DiI concentrations were 1:2000 (mol/mol) relative to bulk lipict. fact be greater tl~an implied in these results, since the positive charges on the indocarbocyanine headgroups and in the N-terminal region of SP-C may repel each other. The concentration of C, DiI in these ET experiments was 1:2000 relative to bulk lipid, low enough to avoid the self-quenching which occurs at higher concentrations of C, DiI. The fluorescence of indocarbocyanine dyes in SL lipids in the presence of 0.5% (unlabeled) SP-C is shown in Fig. 4. With decreasing temperature, the fluorescence of all four C, Dils increased in a gradual manner. A possible confounding factor in ET experiments would be a large change in absorbance of the C, DiI, producing a change in R o, which could cause a change in ET. A change in absorbance would also alter fluorescence intensity. The gradual changes in fluorescence intensity visible in Fig. 4 are similar for the four dyes, and too small to account for the large difference in ET seen in Fig. 3. The RoS for ET from NBD-SP-C to C~2DiI and C~6DiI were calculated from the spectral characteristics of the probes in the SL lipid mixture. Ro, the distance at which ET efficiency is 50%, depends on the fluorescence quantum yield of the

33

donor. The quantum yield of NBD-SP-C increased with decreasing temperature, and most of the increase occurred in the region of the lipid phase transition. For this reason, Ros for each donor/acceptor pair were calculated at both 20 ° and 50°C (Table 1). The Ros for each donor/acceptor pair at 50°C differed from those at 20°C by less than 10%, and therefore could not account for the large difference in ET between the two temperatures. ET from NBD-SP-C to C~2DiI and C16DiI was determined as a function of the acceptor (C, DiI) concentration at 20 ° and 50 ° in SL lipids (Fig. 5). In Fig. 5, ET is plotted as a function of the number of acceptors/Ro 2. The concentration unit acceptors/Ro 2 removes the dependence on Ro from the ET vs. concentration curve and is the 'natural' unit for plotting ET against concentration [38]. At 50°C SP-C is not aggregated [12], and SL lipids are in the fluid phase. The ET observed at 50°C should depend solely on the density of acceptors per Ro E and on the distance of closest approach of donor and acceptor [38]. If the local concentration of C, DiI within the SP-C aggregates were the same at 20°C and 50°C, the ET curves at 20°C and 50°C should overlap. In Fig. 5, the curve of ET from NBD-SP-C to C]2DiI at 20°C is significantly higher than that measured at 50°C, reflecting a higher concentration of C12DiI in the vicinity of SP-C at 20°C than at 50°C. For C~6DiI the 20°C curve is lower than that measured at 50°C, reflecting a lower concentration of Cx6DiI within the SP-C aggregates at 20°C. The 50°C curves for the two probes are close to identical. Because the curves plotted in Fig. 5 are independent of Ro, it is clear that the differences in ET between 20 ° and 50°C cannot be attributed to differences in the Ros at these two temperatures. Unlike SP-C, the hydrophobic surfactant protein SP-B does not appear to penetrate the lipid bilayer, but lies at the bilayer surface, with its more hydrophilic residues exposed to solution. ET from NBD-SP-B to C, DiI in 7:1 DPPC/ D P P G vesicles is shown in Fig. 6. The curves of ET from NBD-SP-B to the various C, DiI are qualitatively similar; all increase in ET near the phase transition. Below 34°C, ET from NBD-SPB to all four probes increased, similar to that of

34

A.D. Horowitz / Chemistry and Physics of Lipids 76 (1995) 27-39 4

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Temperature (*C) Temperature (*C) Fig. 4. Fluorescenceof C, DiI in SL lipid vesicles. Fluorescenceof C, DiI was measured at 565 nm, with excitationat 520 nm. SP-C was present at 0.5% protein/lipid (w/w), or 1:1100(mol/mol) and the C, DiI concentrationswere 1:2000 (mol/mol) relative to bulk lipid. Panel A: R, C12Dil; panel B: T, C16DiI; panel C: A, Ag:2C18DiI;panel D: II, ClsDiI. NBD-SP-C. Slightly greater increases in ET from NBD-SP-B to C12DiI and A9'~2CI8DiI than to CI6DiI and C~8DiI between 37°C and 42°C could arise from lipid disorganization or packing defects in the region of SP-B at the lipid phase transition. ET from NBD-SP-B to the C, DiI was observed in SL lipids (Fig. 7). In contrast to NBD-SP-C, the changes in ET observed with NBD-SP-B were small. Between 39°C and 30°C, slightly greater ET from NBD-SP-B to C16DiI and C~sDiI was observed than to C~2DiI and A9'I2C18DiI. These Table 1 Calculated RoS for energy transfer Donor

Acceptor

20°C

50°C

NBD-SP-C NBD-SP-C NBD-SP-B NBD-SP-B

CI2DiI CI6DiI Ci2DiI C16DiI

66 A 68 ~ 46 A 47 A

63 A 63 A 43 A 43 A

results exclude any preference of SP-B for shorter chain or unsaturated lipids. 4. D i s c u s s i o n

Use of fluorescence energy transfer from labeled proteins to lipid probes which differ only in their acyl chain composition allows direct examination of the lipids in immediate proximity of the hydrophobic surfactant proteins. NBD-SP-C displays greater ET to C~2DiI and A9,12CIsDiI below the bulk phase transition in the SL lipid mixture, but decreased ET to C16DiI and C~sDiI. These observations indicate that SP-C is associated with shorter chain and unsaturated lipids below the D P P C / D P P G phase transition temperature. The SL lipid mixture contains a high proportion of palmitoyl lipids, as does pulmonary surfactant. Below 37°C, SP-C forms aggregates in SL lipids [12]. Decreased ET to C~6DiI and CIsDiI below the phase transition is consistent with exclusion of

35

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Acceptors/R, 2 Fig. 5. Energy transfer from NBD-SP-C to C,2DiI and C~6DiI as a function of temperature and C, DiI concentration. CnDiI concentration in the lipid bilayer is expressedas the number of acceptors (C, DiI molecules) per Ro2. Acceptors/Ro2 is calculated from the number of acceptors per phospholipid, assuming a surface area of 70/It2 per phospholipid and using the Ro values in Table 1. Data were collected at 20°C and 50°C. V, C,2DiI (20°C); 0, C]2DiI (50°C); A, Ci6DiI (20°C) and III, Cl6DiI (50°C).

Like SP-C, the transmembrane proteins bacteriorhodopsin and the photosynthetic reaction center protein of Rhodopseudomonas sphaeroides aggregate in gel phase DPPC bilayer vesicles, but remain dispersed in the fluid phase [39,40]. The transmembrane hydrophobic domain of each of these proteins was estimated to be approximately 30 A [41,40], close to that of SP-C. ET from C,sDII and octadecylrhodamine B to the bacteriorhodopsin chromophore demonstrated exclusion of C,sDiI and octadecylrhodamine B from bacteriorhodopsin aggregates below the lipid phase transition [39]. The model peptide, lysz-gly-leu24lys2-ala-amide (P24), resembles the hydrophobic a-helical region of SP-C: the length of the hydrophobic a-helix of P24 is 24 residues, forming a 0.4 A

0.3

I--.

r" ILl

C16DiI and C18DiI from the aggregates formed by SP-C at low temperatures. As a membrane-spanning protein, the interactions of SP-C with the lipid bilayer are likely to be primarily governed by interaction with the hydrophobic region of the bilayer. The behavior of membrane-spanning proteins has been explained in terms of the degree of mismatch between the length of the protein hydrophobic region and the thickness of the hydrophobic region of the bilayer [28]. Where mismatch occurs, the lipid bilayer and, to a lesser extent, the protein will deform to reduce the exposure of hydrophobic residues to the aqueous phase. Rigid, gel phase bilayers favor the segregation of proteins into separate phases in the bilayer both because of their rigidity, and because the hydrophobic length of gel phase bilayers is longer than that of the same bilayer in the fluid phase,. For DPPC, the hydrophobic length in the gel phase has been calculated to be 39.4 ,~, and 26.3 A in the fluid phase [26].

q\ \\

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0.1

0.0

I

I

30

40

I

50 B

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•

ill C lad

I

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I

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40

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Temperature (°C) Fig. 6. Energy transfer from NBD-SP-B to C, Dil in 7:1 DPPC/DPPG. The NBD-SP-B concentration was maintained at 0.75% protein/lipid (w/w), or 1:1600 (mol/mol). Symbols indicate ET from NBD-SP-B to, in panel A: A, A9'I2ClsDil; fT, C~2DiI; in panel B: I?, C~6DiI; II, CisDiI.

A.D. Horowitz / Chemistry and Physics of Lipids 76 (1995) 27-39

36 0.4

i

A

t,

0.3

tO

i-

0.2

t-hi

0.1

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I

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I 50

B 0.3 ¢-

o r-

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>,.

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I

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Temperature (*C) Fig. 7. Energy transfer from NBD-SP-B to C, DiI in SL lipid vesicles. The NBD-SP-B concentration was maintained at 0.75% protein/lipid (w/w), or 1:1600 (mol/mol) and the C, Dil concentrations were 1:2000 (mol/mol) relative to bulk lipid. Symbols indicate ET from NBD-SP-B to, in panel A: A, Ag,12CIsDiI; Fq, C12DiI; in panel B: T, CI6DiI; II, CisDiI.

hydrophobic region of 31-32 h [26] and the length of the hydrophobic region of the a-helix of SP-C is 23 amino acids, approximately the same as that of P24. At low to moderate peptide concentrations differential scanning calorimetry data of bilayers containing P24 were compatible with a macroscopic mixture of peptide-rich and peptidepoor domains [26]. The transition temperature was decreased and the transition was broadened, similar to the effect of SP-C on DMPC bilayers [24]. Thus, the behavior of SP-C in a DPPC/ DPPG bilayers is consistent with that of similar transmembrane proteins and with predictions of the theory of hydrophobic mismatch. No difference in association of SP-C with short and long

chain lipids is expected above the DPPC/DPPG phase transition temperature, because the hydrophobic length of the lipids in the fluid phase is shorter. Below the DPPC/DPPG phase transition temperature in SL lipids SP-C forms aggregates [12]. The present results show that the aggregates are associated with shorter chain and unsaturated lipids, and exclude long chain saturated lipids. Both SP-C and the shorter chain and'unsaturated lipids are likely to be excluded from the gel phase palmitoyl lipids. The increase in the association of SP-C and short chain lipids at lower temperatures may arise from exclusion of SP-C and shorter chain lipids by the gel phase lipids. In addition, the concentration of shorter chain and unsaturated lipids within SP-C aggregates are likley to be higher for two reasons. First, the hydrophobic length of the shorter chain and unsaturated lipids will be closer to the length of the hydrophobic region of the SP-C a-helix. Secondly, SP-C will cause disordering of lipid acyl chains both due to any 'roughness' of the protein surface in the hydrophobic region, and to deformations of the lipid bilayer to accommodate the shorter hydrophobic region of SP-C. The disorder of the acyl chains will produce defects which will accommodate shorter chain and unsaturated lipids more easily than the highly-ordered gel phase. Since pulmonary surfactant exists in the alveoli in bilayer forms as well as in a monolayer, the behavior of SP-C and SP-B in lipid bilayer vesicles is directly relevant to its biological role. Pulmonary surfactant contains a high proportion of dipalmitoyl lipids. The results presented here demonstrate that SP-C in pulmonary surfactant bilayers will be excluded from gel phase palmitoyl lipids. Although the experiments reported here do not bear directly on the behavior of SP-C in a monolayer, they are consistent with the behavior of SP-C determined by epifluorescence of C12-NBDPC in a DPPC monolayer [42]. SP-C perturbed lipid packing and inhibited lipid condensation at 24°C. High concentrations of SP-C greatly reduced the number and total area of condensed domains. Circular dichroism of SP-C/lipid monolayers which have been deposited on glass slides suggest that SP-C retains its high a-helical con-

A.D. Horowitz / Chemistry and Physics of Lipids 76 (1995) 27-39

tent in the monolayer, and that the axis of the ~-helix m a y be oriented parallel to the air-water interface [43], in which case, the diameter of the SP-C or-helix would be parallel to and shorter than the lipid acyl chains. In contrast to SP-C, ET from NBD-SP-B to CnDiI demonstrated little acyl chain preference on the part of SP-B. This result is consistent with the fact that SP-B is not a membrane-spanning protein. The behavior of SP-B in a lipid bilayer is more likely to be governed by interactions with the lipid headgroup region than by deformations of the lipid acyll chain region arising from hydrophobic mismatch. Due to their differing interactions with lipid,;, SP-C and SP-B may not interact directly in a lipid bilayer. A recent study of the behavior of SP-C and SP-B in D P P C / D P P G monolayers also suggests that they m a y not interact in a monolayer [44]. In conclusion, SP-C aggregates in lipid mixtures containing gel phase D P P C / D P P G are enriched in shorter chain and unsaturated lipids. In view of recent work with model peptides resembling SP-C in secondary structure, the aggregation of SP-C in the presence of gel phase lipids is likely to arise from a mismatch in the length of the hydrophobic region of SP-C relative to D P P C and D P P G . This conclusion is supported by recent structural analysis of SP-C in organic solvents, which revealed a hydrophobic ~-helix much shorter than the hydrophobic region of a gel phase lipid bilayer [16]. SP-B, which is not a membrane-spanning protein, shows little or 11o differential association with any of the acyl chains tested. Use of the indocarbocyanine dyes represents a convenient method to determine lipid acyl chain preferences, since CnDiI with a variety of acyl chain compositions are available. Aggregation of SP-C in lipid bilayers may form the basis for its surface activity, providing a region of destabilized lipid structure which facilitates fusion of the bilayer with a monolayer at the air/water interface. In a monolayer, SP-C appears to pa~:ition into fluid domains [42]. Disruption of lipid domains by SP-C m a y increase surface activity of lipid mixtures by facilitating rapid respreading of the monolayer at the air/liquid interface.

37

Acknowledgments This work was supported by a research grant from the American Lung Association and HL07527. The author thanks Eun D. H a n for excellent technical assistance.

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rearrangements of dipalmitoylphosphatidylcholine in spread monolayers. Biophys. J. 63, 197-204. [431 M.A. Oosterlaken-Dijksterhuis, H.P. Haagsman, L.M.G. van Golde and R.A. Demel (1991) Characterization of lipid insertion into monomolecular layers mediated by lung surfactant proteins SP-B and SP-C. Biochemistry 30, 10965-1097 h [441 S. Taneva and K.M.W. Keough (1994) Pulmonary surfactant proteins SP-B and SP-C in spread monolayers at the air-water interface. III. Proteins SP-B plus SP-C with phospholipids in spread monolayers. Biophys. J. 66, 1158-1166.