Analysis of binding and membrane destabilization of phospholipid membranes by surfactant apoprotein B.
To further elucidate the nature of the molecular interactions of surfactant apoprotein B (SP-B) with phospholipid (PL) membranes, we studied the binding of SP-B to PL membranes and the lipid-dependency of its subsequent effects on leakage and fusion of membranes. SP-B binding to membranes was studied by labeling the protein with the fluorophore 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) and measuring the fluorescence of the labeled protein in the presence of varying amounts of dipalmitoylphosphatidylcholine-egg phosphatidylglycerol (DPPC-eggPG; 7-3). Leakage of contents from liposomes made of DPPC and varying molar fraction of egg phosphatidylcholine (eggPC) or eggPG was assessed by measuring the fluorescence of entrapped water-soluble probes ANTS and DPX. Fusion of membranes was assessed by measuring the fluorescence of membrane-bound NBD-phosphatidylethanolamine (NBD-PE) and rhodamine-PE (RHO-PE). We found that SP-B bound to PL membranes with high affinity and appeared to irreversibly cluster at the membrane surface, leading to graded release of the vesicle contents and eventually fusion of the membranes with increasing protein-lipid ratios. All lipid mixtures tested were susceptible to the membrane disruptive effects of SP-B, but DPPC-eggPG membranes displayed a biphasic response to increasing molar fractions of eggPG, whereas increasing fractions of eggPC elicited a monotonic response.
Chang
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Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA 94143-1245, USA.
Nir
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Poulain
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D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism*; D002645:Chickens; D004285:Dogs; D006801:Humans; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008561:Membrane Fusion; D008567:Membranes, Artificial; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000737:chemistry / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000737:chemistry* / Q000378:metabolism*
D001059:Apoproteins; D008081:Liposomes; D008567:Membranes, Artificial; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D010743:Phospholipids; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; C046616:pulmonary surfactant apoprotein