Surface study of apoB1694–1880, a sequence that can anchor apoB to lipoproteins and make it nonexchangeable
2009; Elsevier BV; Volume: 50; Issue: 7 Linguagem: Inglês
10.1194/jlr.m900040-jlr200
ISSN1539-7262
AutoresLibo Wang, Dale D. O. Martin, E.I. Genter, Jianjun Wang, Roger S. McLeod, Donald Small,
Tópico(s)Lipid metabolism and disorders
ResumoApolipoprotein B (apoB) is a nonexchangeable apolipoprotein. During lipoprotein assembly, it recruits phospholipids and triacylglycerols (TAG) into TAG-rich lipoprotein particles. It remains bound to secreted lipoproteins during lipid metabolism in plasma. The β1 region (residues 827–1880) of apoB has a high amphipathic β strand (AβS) content and is proposed to be one region anchoring apoB to lipoproteins. The AβS-rich region between apoB37 and apoB41 (residues 1694–1880) was cloned, expressed, and purified. The interfacial properties were studied at the triolein/water (TO/W) and air/water (A/W) interfaces. ApoB[37–41] is surface-active and adsorbs to the TO/W interface. After adsorption the unbound apoB[37–41] was removed from the aqueous phase. Adsorbed apoB[37–41] did not desorb and could not be forced off by increasing the surface pressure up to 23 mN/m. ApoB[37–41] adsorbed on the TO/W interface was completely elastic when compressed and expanded by ±13% of its area. On an A/W interface, the apoB[37–41] monolayer became solid when compressed to 4 mN/m pressure indicating extended β-sheet formation. It could be reversibly compressed and expanded between low pressure and its collapse pressure (35 mN/m). Our studies confirm that the AβS structure of apoB[37–41] is a lipid-binding motif that can irreversibly anchor apoB to lipoproteins. Apolipoprotein B (apoB) is a nonexchangeable apolipoprotein. During lipoprotein assembly, it recruits phospholipids and triacylglycerols (TAG) into TAG-rich lipoprotein particles. It remains bound to secreted lipoproteins during lipid metabolism in plasma. The β1 region (residues 827–1880) of apoB has a high amphipathic β strand (AβS) content and is proposed to be one region anchoring apoB to lipoproteins. The AβS-rich region between apoB37 and apoB41 (residues 1694–1880) was cloned, expressed, and purified. The interfacial properties were studied at the triolein/water (TO/W) and air/water (A/W) interfaces. ApoB[37–41] is surface-active and adsorbs to the TO/W interface. After adsorption the unbound apoB[37–41] was removed from the aqueous phase. Adsorbed apoB[37–41] did not desorb and could not be forced off by increasing the surface pressure up to 23 mN/m. ApoB[37–41] adsorbed on the TO/W interface was completely elastic when compressed and expanded by ±13% of its area. On an A/W interface, the apoB[37–41] monolayer became solid when compressed to 4 mN/m pressure indicating extended β-sheet formation. It could be reversibly compressed and expanded between low pressure and its collapse pressure (35 mN/m). Our studies confirm that the AβS structure of apoB[37–41] is a lipid-binding motif that can irreversibly anchor apoB to lipoproteins. Apolipoprotein B (apoB) is a large protein (4536 residues) that plays an essential role in the formation of triacylglycerol (TAG)-rich lipoproteins by the intestine, as chylomicrons, or by the liver, as VLDL (1Kane J.P. Havel R.J. Disorders of the biogenesis and secretion of lipoproteins containing the B apolipoproteins.in: Scriver C.R. Beaudet A.L. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw–Hill, New York2001: 2717-2752Google Scholar). The N-terminal 48% of apoB (apoB48) in the intestine and the full-length apoB (apoB100) in the liver play fundamental roles in the assembly with lipids, including phosphatidylcholine, TAG, and cholesterol, into a nascent emulsion particle which, after further modification, is secreted and ultimately enters the blood. These particles carry dietary (chylomicrons) or liver (VLDL) TAG through the blood to other tissues where they are acted upon by lipoprotein lipase to serve as a source of energy or for cell membrane and lipid droplet synthesis. ApoB is unique among apolipoproteins and a rare member of the family of proteins that bind irreversibly to lipid droplets (1Kane J.P. Havel R.J. Disorders of the biogenesis and secretion of lipoproteins containing the B apolipoproteins.in: Scriver C.R. Beaudet A.L. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw–Hill, New York2001: 2717-2752Google Scholar, 2Havel R.J. Kane J.P. Introduction: structure and metabolism of plasma lipoproteins.in: Scriver C.R. Beaudet A.L. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw–Hill, New York2001: 2705-2716Google Scholar). Only a few other peptides share this irreversible binding, including the oleosins of oil bodies in seeds (3Huang A.H.C. Oleosins and oil bodies in seeds and other organs.Plant Physiol. 1996; 110: 1055-1061Crossref PubMed Scopus (435) Google Scholar) and perhaps some viral core proteins such as those in hepatitis C virus (4Hope R.G. Murphy D.J. McLauchlan J. The domains required to direct core proteins of hepatitis C virus and GB virus-B to lipid droplets share common features with plant oleosin proteins.J. Biol. Chem. 2002; 277: 4261-4270Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Oleosins and virus core proteins have a large hydrophobic, proline-rich region that has been suggested to anchor these peptides permanently to their respective emulsion particles. ApoB is the only nonexchangeable apolipoprotein: it remains with the particle from the time it is formed in the liver or intestine until it is removed and catabolized through receptor-mediated cell uptake (1Kane J.P. Havel R.J. Disorders of the biogenesis and secretion of lipoproteins containing the B apolipoproteins.in: Scriver C.R. Beaudet A.L. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw–Hill, New York2001: 2717-2752Google Scholar, 2Havel R.J. Kane J.P. Introduction: structure and metabolism of plasma lipoproteins.in: Scriver C.R. Beaudet A.L. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw–Hill, New York2001: 2705-2716Google Scholar). All remaining plasma apolipoproteins are exchangeable, moving between different lipoproteins as they circulate and are remodeled in the plasma. ApoB100 has been shown to bind to TAG droplets and to undergo conformational changes in response to changes in surface pressure (5Wang L. Walsh M.T. Small D.M. Apolipoprotein B is conformationally flexible but anchored at a triolein/water interface: a possible model for lipoprotein surfaces.Proc. Natl. Acad. Sci. USA. 2006; 103: 6871-6876Crossref PubMed Scopus (46) Google Scholar). While the peptide cannot be completely displaced from the triolein/water (TO/W) interface, parts of the peptide appear to desorb as surface pressure is increased and then to readsorb rapidly as the pressure is reduced (5Wang L. Walsh M.T. Small D.M. Apolipoprotein B is conformationally flexible but anchored at a triolein/water interface: a possible model for lipoprotein surfaces.Proc. Natl. Acad. Sci. USA. 2006; 103: 6871-6876Crossref PubMed Scopus (46) Google Scholar). Previous studies have shown that a 12 amino acid consensus amphipathic β strand (AβS) or a two-strand amphipathic β sheet modeled from B21 to B41 sequence (the first β sheet region of apoB) binds irreversibly to air/water (A/W), hydrocarbon/water (HC/W), and TO/W interfaces, lowering the interfacial free energy (6Wang L. Small D.M. Interfacial properties of amphipathic β strand consensus peptides of apolipoprotein B at oil/water interfaces.J. Lipid Res. 2004; 45: 1704-1715Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Compression to high surface pressure does not displace the peptide from the interface but simply compresses it. These AβS peptides form an almost completely elastic interface at the A/W, HC/W, and TO/W surfaces. From predictions of secondary structure and studies on consensus AβS peptides, it was suggested that the β1 (residues 827–1880) and β2 (residues 2571–4000) domains of apoB100 are the anchors that prevent apoB from leaving the particle once it is assembled (5Wang L. Walsh M.T. Small D.M. Apolipoprotein B is conformationally flexible but anchored at a triolein/water interface: a possible model for lipoprotein surfaces.Proc. Natl. Acad. Sci. USA. 2006; 103: 6871-6876Crossref PubMed Scopus (46) Google Scholar, 6Wang L. Small D.M. Interfacial properties of amphipathic β strand consensus peptides of apolipoprotein B at oil/water interfaces.J. Lipid Res. 2004; 45: 1704-1715Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 7Segrest J.P. Jones M.K. De Loof H. Dashti N. Structure of apolipoprotein B-100 in low density lipoproteins.J. Lipid Res. 2001; 42: 1346-1367Abstract Full Text Full Text PDF PubMed Google Scholar–8Small D.M. Atkinson D. The first beta sheet region of apoB(apoB21–41) is a amphipathic ribbon 50–60Å wide and 200Å long which initiates triglyceride binding and assembly of nascent lipoproteins.Circulation. 1997; 96: I-1Google Scholar). ApoB48 of course has only the β1 putative anchoring domain. Based on a consensus of several secondary structure modeling algorithms, the sequence of apoB from B21 to B41 (residues 968–1880) was originally suggested to be a relatively continuous large, amphipathic β sheet roughly 50–60 Å in width and about 200 Å long (8Small D.M. Atkinson D. The first beta sheet region of apoB(apoB21–41) is a amphipathic ribbon 50–60Å wide and 200Å long which initiates triglyceride binding and assembly of nascent lipoproteins.Circulation. 1997; 96: I-1Google Scholar). It is known that the region between B19.5 and B22 can initiate the formation of lipoprotein particles that contain phospholipids and some TAGs in various cell lines (9Yao Z.M. Blackhart B.D. Linton M.F. Taylor S.M. Young S.G. McCarthy B.J. Expression of carboxyl-terminally truncated forms of human apolipoprotein B in rat hepatoma cells. Evidence that the length of apolipoprotein B has a major effect on the buoyant density of the secreted lipoproteins.J. Biol. Chem. 1991; 266: 3300-3308Abstract Full Text PDF PubMed Google Scholar, 10Spring D.J. Chen-Liu L.W. Chatterton J.E. Elovson J. Schumaker V.N. Lipoprotein assembly. Apolipoprotein B size determines lipoprotein core circumference.J. Biol. Chem. 1992; 267: 14839-14845Abstract Full Text PDF PubMed Google Scholar, 11McLeod R.S. Wang Y. Wang S. Rusiñol A. Links P. Yao Z. Apolipoprotein B sequence requirements for hepatic very low density lipoprotein assembly. Evidence that hydrophobic sequences within apolipoprotein B48 mediate lipid recruitment.J. Biol. Chem. 1996; 271: 18445-18455Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 12Carraway M. Herscovitz H. Zannis V. Small D.M. Specificity of lipid incorporation is determined by sequences in the N-terminal 37 of apoB.Biochemistry. 2000; 39: 9737-9745Crossref PubMed Scopus (28) Google Scholar, 13Shelness G.S. Hou L. Ledford A.S. Parks J.S. Weinberg R.B. Identification of the lipoprotein initiating domain of apolipoprotein B.J. Biol. Chem. 2003; 278: 44702-44707Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 14Manchekar M. Richardson P.E. Forte T.M. Datta G. Segrest J.P. Dashti N. Apolipoprotein B-containing lipoprotein particle assembly: lipid capacity of the nascent lipoprotein particle.J. Biol. Chem. 2004; 279: 39757-39766Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 15Richardson P.E. Manchekar M. Dashti N. Jones M.K. Beigneux A. Young S.G. Harvey S.C. Segrest J.P. Assembly of lipoprotein particles containing apolipoprotein-B: structural model for the nascent lipoprotein particle.Biophys. J. 2005; 88: 2789-2800Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar–16Dashti N. Manchekar M. Liu Y. Sun Z. Segrest J.P. Microsomal triglyceride transfer protein activity is not required for the initiation of apolipoprotein B-containing lipoprotein assembly in McA-RH7777 cells.J. Biol. Chem. 2007; 282: 28597-28608Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). In C-127 cells containing very low levels of the catalyst microsomal triglyceride transfer protein (MTP), lipoproteins secreted from cells transfected with N-terminal fragments of apoB show an abrupt increase in TAG secretion when the secreted apoB polypeptide is longer than the N-terminal B32. The number of TAG molecules increases from 25 in secreted B32 particles to 113 in B37 particles and to 191 in B41 particles (12Carraway M. Herscovitz H. Zannis V. Small D.M. Specificity of lipid incorporation is determined by sequences in the N-terminal 37 of apoB.Biochemistry. 2000; 39: 9737-9745Crossref PubMed Scopus (28) Google Scholar). B37 and B41 nascent particles are quasi-spherical with a TAG core clearly observable by cryoelectron microscopy. Thus, this part of the β1 region of apoB (B32–B41) appears to favor the recruitment of TAG, presumably by binding nascent TAG as the sequence translocates through the endoplasmic reticulum. Having shown that small AβS bind irreversibly, we needed to show that an actual sequence from apoB, predicted to be rich in AβS and associated with the secretion of TAG, has properties similar to the small consensus peptides (6Wang L. Small D.M. Interfacial properties of amphipathic β strand consensus peptides of apolipoprotein B at oil/water interfaces.J. Lipid Res. 2004; 45: 1704-1715Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). We cloned, expressed, and purified a polypeptide from the β strand–rich region between apoB37 and 41 (amino acids 1694–1880) and then studied its behavior at the oil/water and A/W interfaces. This region contains 10 AβS of 11–15 amino acids (Fig. 1) and several shorter strands (7Segrest J.P. Jones M.K. De Loof H. Dashti N. Structure of apolipoprotein B-100 in low density lipoproteins.J. Lipid Res. 2001; 42: 1346-1367Abstract Full Text Full Text PDF PubMed Google Scholar, 8Small D.M. Atkinson D. The first beta sheet region of apoB(apoB21–41) is a amphipathic ribbon 50–60Å wide and 200Å long which initiates triglyceride binding and assembly of nascent lipoproteins.Circulation. 1997; 96: I-1Google Scholar). The total region is at least 70% β strand. Studies of this sequence show that it is irreversibly bound to the TAG/water interface; it cannot be pushed off by Π up to 24 mN/m; and it is almost completely elastic, therefore a potential anchoring region of apoB to triaclyglycerol-rich lipoprotein. This study provides substantial validity to the previous speculations, derived from small peptide studies, suggesting that AβS regions of apoB serve to anchor apoB48 and apoB100 to their lipoproteins. A fragment encompassing codons for apoB1694–1880 (the region from B37 to B41, apoB[37Read J. Anderson T.A. Ritchie P.J. Vanloo B. Amey J. Levitt D. Rosseneu M. Scott J. Shoulders C.C. A mechanism of membrane neutral lipid acquisition by the microsomal triglyceride transfer protein.J. Biol. Chem. 2000; 275: 30372-30377Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 38Stillemark P. Borén J. Andersson M. Larsson T. Rustaeus S. Karlsson K.A. Olofsson S.O. The assembly and secretion of apolipoprotein B-48-containing very low density lipoproteins in McA-RH7777 cells.J. Biol. Chem. 2000; 275: 10506-10513Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 39Herscovitz H. Hadzopoulou-Cladaras M. Walsh M.T. Cladaras C. Zannis V.I. Small D.M. Expression, secretion, and lipid-binding characterization of the N-terminal 17% of apolipoprotein B.Proc. Natl. Acad. Sci. USA. 1991; 88: 7313-7317Crossref PubMed Scopus (50) Google Scholar, 40Herscovitz H. Derksen A. Walsh M.T. McKnight C.J. Gantz D.L. Hadzopoulou-Cladaras M. Zannis V. Curry C. Small D.M. The N-terminal 17% of apoB binds tightly and irreversibly to emulsions modeling nascent very low density lipoproteins.J. Lipid Res. 2001; 42: 51-59Abstract Full Text Full Text PDF PubMed Google Scholar, 41Weinberg R.B. Cook V.R. DeLozier J.A. Shelness G.S. Dynamic interfacial properties of human apolipoproteins A-IV and B-17 at the air/water and oil/water interface.J. Lipid Res. 2000; 41: 1419-1427Abstract Full Text Full Text PDF PubMed Google Scholar]) from the apoB48 cDNA (17Hussain M.M. Zhao Y. Kancha R.K. Blackhart B.D. Yao Z. Characterization of recombinant human apoB-48–containing lipoproteins in rat hepatoma McA-RH7777 cells transfected with apoB-48 cDNA: overexpression of apoB-48 decreases synthesis of endogenous apoB-100.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 485-494Crossref PubMed Scopus (63) Google Scholar) was subcloned using primers containing NcoI and HindIII restriction sites for the in-frame insertion of apoB sequence immediately downstream of a His6-Ser-Ser- tag sequence in a modified pET30a plasmid (pET30a_sHT) (pETB37sense: 5′-TCTAAGGCCATGGTCGACAGCAAAAACATTTTC-3′; pETB41anti: 5′-CGGCCGCAAGCTTAGATGGTCATGGTAAACGG-3′). Following amplification of the fragment using Vent DNA polymerase (New England Biolabs, Boston), the amplified fragment was digested with NcoI and HindIII and ligated to pET30a_sHT which had been digested with the same enzymes. Transformed E. coli (BL21) were selected on kanamycin (30 μg/ml) and screened for insert. Bacteria strain BL21(DE3) was transformed with pETB3741 plasmid, and a single colony was selected and transferred to 3 mls of LB-kanamycin broth and grown at 37°C to A600 of approximately 1.0. The bacteria were collected by centrifugation and transferred to 100 ml of fresh LB-kanamycin broth and grown to A600 of approximately 1.0. The vigorous culture was finally transferred to 900 mls of LB-kanamycin and grown to an A600 of 0.6–0.8. Isopropyl-thioglucoside (IPTG) was added to a final concentration of 0.3 mM, and the incubation was continued for 2.5 h at 37°C. The bacteria were then collected by centrifugation, and the pellet was stored at −80°C. To purify the apoB1694–1880 fragment, the bacteria were thawed on ice and suspended in 20 ml of 50 mM sodium phosphate, pH 8.0, 150 mM NaCl containing one-half of a protease inhibitor tablet (Complete®, Roche) and 5 ml of lysozyme solution (4 mg/ml). The suspension was then sonicated (6 × 10 s), and the inclusion bodies containing the apoB protein were collected by centrifugation (30 min, 10000 rpm, SS34 rotor). Inclusion bodies were solubilized in 25 ml of 50 mM sodium phosphate, pH 8.0, 150 mM NaCl, 8 M urea, and protease inhibitors. After solubilization, the solution was mixed with a 50% (v/v) suspension of Ni-NTA His-Bind resin (Novagen) for 1 h. The resin was poured into a disposable chromatography column and washed successively with 50 mM sodium phosphate, 8 M urea at pH 8.0 (10 ml), and then at pH 5.9 (20 ml). ApoB1694–1880 was then eluted from the column with 10 ml of 50 mM sodium phosphate, pH 4.3, 8 M urea and dialyzed against 2 l of 20 mM sodium phosphate, pH 3.0, 50 mM NaCl. For storage and transport, the protein was dialyzed against 5 mM acetic acid and lyophilized. The purification profile is shown in Fig. 2. Circular dichroism (CD) spectra were collected between 190 and 260 nm on an OLIS DSM CD spectrophotometer at 25°C in 20 mM phosphate buffer containing 50 mM NaCl. In pH experiments, the pH was adjusted by changing the ratios of monobasic and dibasic sodium phosphate and verified by pH measurements. Crosslinking studies were performed on 84 μM samples at low pH after overnight incubation as previously described (18Ren X. Zhao L. Sivashanmugam A. Miao Y. Korando L. Yang Z. Reardon C.A. Getz G. Brouillette C.G. Jerome W.G. et al.Engineering mouse apolipoprotein A-I into a monomeric, active protein useful for structural determination.Biochemistry. 2005; 44: 14907-14919Crossref PubMed Scopus (17) Google Scholar). The interfacial tension of the TO/W interface in the presence of apoB[37–41] peptide in the aqueous phase was measured with an I. T. CONCEPT (Longessaigne, France) Tracker oil-drop tensiometer (19Labourdenne S. Gaudry-Rolland N. Letellier S. Lin M. Cagna A. Esposito G. Verger R. Rivière C. The oil-drop tensiometer: potential applications for studying the kinetics of (phopho)lipase action.Chem. Phys. Lipids. 1994; 71: 163-173Crossref Scopus (91) Google Scholar). An aliquot of peptide stock (approximately 0.1–0.6 mg/ml apoB[37–41] dissolved in 2 mM pH 3.0 phosphate buffer) was added to the aqueous phase (2 mM pH 4.85 phosphate buffer) to obtain a peptide concentrations ranging from 2.6 × 10−8 M to 2.2 × 10−7 M. At these concentrations and pH 4.85, apoB[37–41] peptide is soluble. A 16 μl triolein drop was formed in the aqueous phase and the interfacial tension (γ) was recorded continuously until it approached an equilibrium level. The surface pressure Π was obtained from γ of the interface without peptide (γTO) minus γ of the interface with peptide (γpep) (i.e., Π = γTO − γpep). All experiments were carried out at 25 ± 0.1°C in a thermostated system. All the peptide stock concentrations were determined by Lowry protein assay. Triolein (greater than 99% pure) was purchased from NU-CHEK PREP, INC. (Elysian, MN);its interfacial tension against buffer was 32 mN/m. All other reagents were of analytical grade. KCl was heated to 600°C for 6 h to remove all organic contaminants before use. While doing the surface tension measurements, we exchanged the aqueous phase buffer (6 ml) containing the peptide with buffer without peptide by continuously removing the aqueous phase from the surface and infusing new buffer continuously near the bottom of the stirred cuvette. Usually approximately 150 ml buffer was exchanged. This exchange volume removed greater than 99% of the peptide in the aqueous phase. If peptide desorbs into the aqueous phase during or after exchange, then surface concentration will fall and γ will rise. Once γ approached an equilibrium level, the oil drop (16 μl) was compressed by rapidly decreasing the volume by as little as 6% (1 μl) or as much as 25% (4 μl). In special cases the volume was decreased by 50% (8 μl). The sudden decrease in drop volume (V) instantaneously decreased the drop surface area (A) and resulted in a sudden compression causing γ to drop abruptly to a certain level, γ0, and generated an instant surface pressure, Π0 = γTO − γ0, where γTO is the surface tension of pure triolein (32 mN/m). The reduced volume was held for several minutes, and γ was continuously recorded. If it rose back toward γeq then ΠMAX, the maximum Π allowed without causing displacement of part of the peptide from the surface was estimated (5Wang L. Walsh M.T. Small D.M. Apolipoprotein B is conformationally flexible but anchored at a triolein/water interface: a possible model for lipoprotein surfaces.Proc. Natl. Acad. Sci. USA. 2006; 103: 6871-6876Crossref PubMed Scopus (46) Google Scholar). Then the surface was expanded by increasing the drop volume back to the original volume (16 μl) and then held constant for several minutes. This procedure was done both before buffer exchange and after buffer exchange. In either case γ will fall after compression. If peptide molecules readily desorbed from the surface, γ would rise back toward an equilibrium value (the desorption curve). If they do not desorb, γ would remain at a relatively low level. When the compressed surface is re-expanded to the initial area, the change in surface tension may depend on whether there is an aqueous concentration of peptide or whether the peptide has been removed. Before peptide removal, if compression caused desorption of the peptide, then re-expansion will provide space for peptide in the aqueous phase to readsorb to the surface. The kinetics of readsorption will be slow, similar to the kinetics of the original adsorption from the aqueous solution. If the peptide compresses but does not desorb, the peptide should rapidly return to γeq on re-expansion. After the peptide has been removed from the aqueous phase, re-expansion after compression could follow two paths. If peptide has been desorbed, the surface tension will rise to a higher value than the original equilibrium γ and remain constant at the high value, because in the absence of peptide in the aqueous phase no readsorption can occur. If, on the other hand, peptide was not desorbed but merely compressed, the peptide will simply re-expand to conform to the larger area, and surface tension will rise back to the original equilibrium value. A third possible change might occur at high pressure where the peptide is compressed into a small area and partly leaves the surface to refold into a denatured conformation. In this case, the peptide might occupy a smaller area and on re-expansion appear to have been lost or very slowly respread, gradually decreasing γ back toward equilibrium. Oscillations were carried out at the equilibrium γ (γe). The drop volume (16 μl) was sinusoidally oscillated at varied amplitudes (6%–25%) and periods (8–128 s) after γ reached an equilibrium level (γe). The area (A) and γ changes were followed as the volume (V) oscillated. In the elasticity analysis, the interfacial elasticity modulus ε (ε = dγ/d ln A), the phase angle φ between compression and expansion, and the elasticity real part ε′ and the elasticity imaginary part ε″ were obtained (ε′ = |ε|cosφ, ε″ = |ε|sinφ) (20Benjamins J. Lucassen-Reynders E.H. Surface dilational rheology of proteins adsorbed at air/water and oil/water interfaces.in: Möbius D. Miller R. Proteins at Liquid Interfaces. Elsevier, Amsterdam1998: 341-384Crossref Scopus (107) Google Scholar, 21Benjamins J. Cagna A. Lucassen-Reynders E.H. Viscoelastic properties of triacylglycerol/water interfaces covered by proteins.Coll. Surf. 1996; 114: 245-254Crossref Scopus (333) Google Scholar). A 16μl drop was formed in a solution containing 1.3 × 10−7 M of peptide. After reaching γeq (approximately 16 mN/m), the peptide solution was exchanged with peptide-free buffer. The drop was then compressed at a rate of 0.02 μl/s to a certain volume. The compressed volume was held for several minutes and then was re-expanded at a rate of 0.02 μl/s to the original volume. After remaining at 16 μl for several minutes, the compression was repeated but at a larger total compression. Compressions were made at 1, 2, 3, 4, 5, 6, and 8 μl giving surface decreases in area of approximately 5, 8, 13, 17, 22, 27, and 34%. Area, volume, and γ were continuously monitored. After the final compression, the area was re-expanded, and γ was monitored for 20 h. A solution of apoB[37–41] peptide (45 μg/ml) in 30% w/v isopropanol/2 mM phosphate buffer (pH 3.0) was spread slowly (approximately 50 μl/min) on a clean surface of 3.5 M KCl 10 mM pH 7.4 PBS buffer at 25°C according to the techniques of Phillips and Krebs (22Phillips M.C. Krebs K.E. Studies of apolipoproteins at the air-water interface.Methods Enzymol. 1986; 128: 387-403Crossref PubMed Scopus (54) Google Scholar). Monolayers of 9 μg apoB[37–41] were compressed at 5 mN/m per minute on a KSV 5000 mini trough of a Langmuir/Pockles surface balance (Helsinki, Finland) and Π-A curves were obtained. To check the reversibility of the Π/A isotherms of the apoB[37–41] monolayer, the monolayer was first compressed to certain Π (15, 30, or 40 mN/m) and then expanded at 5 mN/m per min to reach a Π lower than 1 mN/m, respectively. The state of the monolayer (liquid, condensed viscous, or solid phase) was detected by putting talc powder on the surface, then directing a fine jet of air and directly observing the motion of talc particles (23Fahey D.A. Small D.M. Surface properties of 1,2-dipalmitoyl-3-acyl-sn-glycerols.Biochemistry. 1986; 25: 4468-4472Crossref PubMed Scopus (17) Google Scholar, 24Fahey D.A. Small D.M. Phase behavior of monolayers of 1,2-dipalmitoyl-3-acyl-sn-glycerols.Langmuir. 1988; 4: 589-594Crossref Scopus (14) Google Scholar). In the liquid state, the talc particles move rapidly and freely; in the condensed viscous state, they move slowly; and in the solid state, they are nearly stationary. The apoB[37–41] fragment was soluble up to 8.4 × 10−5 M at low pH and present in solution primarily as monomers although some aggregates were seen in crosslinking experiments. The pH was raised in steps and the far UV CD spectrum was recorded as a function of pH (Fig. 3A). The loss of solubility appeared to coincide with the loss of the characteristic β secondary structure features between 210 and 217 nm. When the molar ellipticity at 214 nm was plotted against pH (Fig. 3B), the apparent loss of features of β-structure were evident above pH 5.5. This apparent loss secondary structure is due to the loss of protein from solution as precipitation occurs. Therefore, subsequent studies were performed at low concentrations of 2.2 × 10−7 M or lower and at pH of 4.85 where the protein is soluble and probably predominantly monomeric. Concentrations of peptide from 2.6 × 10−8 to 2.2 × 10−7 M in the aqueous phase were studied. The lowest concentrations lowered γ to only 30 mN/m (2.6 × 10−8 M) and 21.3 mN/m (5.2 × 10−8 M). In the latter case, equilibration was carried out for 12,000 s. Sixteen individual experiments were carried out at concentrations from 7.9 × 10−8 to 2.2 × 10−7; the mean γeq was 16.3 ± 1.1 mN/m. There was no significant difference between γeq and concentration in these experiments. Eight experiments were carried out at 8.6 × 10−8 M (γeq= 16.2 ± 0.8 mN/m). Most of the data shown were from these experiments except as noted. Figure 4Ashows a typical adsorption curve (0–7200 s) of apoB[37–41] at TO/W interface and the instant compression and expansion of the interface after γ reached an equilibrium level (after 7200 s). The peptide concentration in the aqueous phase is 8.6 × 10−8 M. In the top figure, the left side (0–7200 s) shows that apoB[37–41] peptide is surface-active and lowered the surface tension (γ) of the TO/W interface (32 mN/m) to reach an equilibrium level (γeq) in about 7200 s. The right side (after 7200 s) shows an example of the tension-time curve of instant compression and re-expansion of the interface with 8.6 × 10−8 M apoB[37–41] peptide in the aqueous phase. After γ approached an equilibrium level, the 16 μl triolein droplet was compres
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