Superficial disposition of the N-terminal region of the surfactant proteinSP-C and the absence of specific SP-B-SP-C interactions in phospholipid bilayers

A dansylated form of porcine surfactant-associated protein C (Dns-SP-C), be aring a single dansyl group at its N-terminal end, has been used to charact erize the lipid-protein and protein-protein interactions of SP-C reconstitu ted in phospholipid bilayers, using fluorescence spectroscopy. The fluoresc ence emission spectrum of Dns-SP-C in phospholipid bilayers is similar to t he spectrum of dansyl-phosphatidylethanolamine, and indicates that the N-te rminal end of the protein is located at the surface of the membranes and is exposed to the aqueous environment. In membranes containing phosphatidylgl ycerol (PG), the fluorescence of Dns-SP-C shows a 3-fold increase with resp ect to the fluorescence of phosphatidylcholine (PC), suggesting that electr ostatic lipid-protein interactions induce important effects on the structur e and disposition of the N-terminal segment of the protein in these membran es. This effect saturates above 20% PG molar content in the bilayers. The p arameters for the interaction of Dns-SP-C with PC or PG have been estimated from the changes induced in the fluorescence emission spectrum of the prot ein. The protein had similar K-d values for its interaction with the differ ent phospholipids tested, of the order of a few micromolar. Cooling of Dns- SP-C-containing dipalmitoyl PC bilayers to temperatures below the phase tra nsition of the phospholipid produced a progressive blue-shift of the fluore scence emission of the protein. This effect is interpreted as a consequence of the transfer of the N-terminal segment of the protein into less polar e nvironments that originate during protein lateral segregation. This suggest s that conformation and interactions of the N-terminal segment of SP-C coul d be important in regulating the lateral distribution of the protein in sur factant bilayers and monolayers. Potential SP-B-SP-C interactions have been explored by analysing fluorescence resonance energy transfer (RET) from th e single tryptophan in porcine SP-B to dansyl in Dns-SP-C. RET has been det ected in samples where native SP-B and Dns-SP-C were concurrently reconstit uted in PC or PG bilayers. However, the analysis of the dependence of RET o n the protein density excluded specific SP-B-Dns-SP-C associations.