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

Abstract

A dansylated form of porcine surfactant-associated protein C (Dns-SP-C), bearing a single dansyl group at its N-terminal end, has been used to characterize the lipid-protein and protein-protein interactions of SP-C reconstituted in phospholipid bilayers, using fluorescence spectroscopy. The fluorescence emission spectrum of Dns-SP-C in phospholipid bilayers is similar to the spectrum of dansyl-phosphatidylethanolamine, and indicates that the N-terminal end of the protein is located at the surface of the membranes and is exposed to the aqueous environment. In membranes containing phosphatidylglycerol (PG), the fluorescence of Dns-SP-C shows a 3-fold increase with respect to the fluorescence of phosphatidylcholine (PC), suggesting that electrostatic lipid-protein interactions induce important effects on the structure and disposition of the N-terminal segment of the protein in these membranes. This effect saturates above 20% PG molar content in the bilayers. The parameters 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 protein. The protein had similar K(d) values for its interaction with the different phospholipids tested, of the order of a few micromolar. Cooling of Dns-SP-C-containing dipalmitoyl PC bilayers to temperatures below the phase transition of the phospholipid produced a progressive blue-shift of the fluorescence 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 environments that originate during protein lateral segregation. This suggests that conformation and interactions of the N-terminal segment of SP-C could be important in regulating the lateral distribution of the protein in surfactant bilayers and monolayers. Potential SP-B-SP-C interactions have been explored by analysing fluorescence resonance energy transfer (RET) from the single tryptophan in porcine SP-B to dansyl in Dns-SP-C. RET has been detected in samples where native SP-B and Dns-SP-C were concurrently reconstituted in PC or PG bilayers. However, the analysis of the dependence of RET on the protein density excluded specific SP-B-Dns-SP-C associations.

The N-terminal segment of pulmonary surfactant lipopeptide SP-C has intrinsic propensity to interact with and perturb phospholipid bilayers

“…The importance of an electrostatic component for the lipid-protein interaction of SP-C has been reported previously [15,18,34]. Native SP-C [21], and specifically its N-terminal segment [23], adopts a location and conformation in anionic phospholipids different from that in zwitterionic phospholipids. Positively charged residues in SP-C seem to be essential for the biophysical activity of the protein [17], supporting the idea that the interaction between its N-terminal segment and anionic phospholipid head-groups has a functional significance.…”

“…We propose also that embedding of the N-terminal segment of SP-C in the bilayer surface is probably an important feature in modulating lateral distribution of the protein in surfactant membranes [23].…”

“…A major problem is that most of the physico-chemical features of SP-C are dominated by contributions from the hydrophobic α-helix, making it difficult to assign relevant properties originating from the N-terminal segment of the protein. Lipidprotein interactions involving this region of SP-C have been studied previously by using protein forms that were chemically derivatized to introduce extrinsic probes at the N-terminal end [22,23]. Those studies indicated that the N-terminal segment of SP-C is located at the surface of phospholipid bilayers, exposed to the aqueous environment.…”

Recent advances in alveolar biology: Evolution and function of alveolar proteins

“…2). Many of the functions of SP-C are attributed to the very dynamic N-terminal domain, which is capable of interacting with and perturbing the lipid packing of phospholipid membranes and monolayer films (Plasencia et al, 2001a,b, 2004, 2005). Specifically, SP-C is able to stabilise the surfactant film during dynamic compression/expansion cycles, a function that is attributed to the palmitoylation of the two cysteine residues in the N-terminal domain of the protein (Qanbar et al, 1996; Gustafsson et al, 2000) (see Fig.…”

Fluorescence as a Tool to Study Lipid-Protein Interactions: The Case of α-Synuclein

“…Two of the most widely used probes, dansyl chloride (DNS-Cl) which stands for 1-dimetylamino-5-naphthylsulfonyl chloride, and fluorescein isothiocyanate (FITC) are shown in Figure 2. These probes react with the free amino groups of proteins, resulting in proteins that fluoresce at blue (DNS) [7] or green (FITC) wavelengths. Proteins can also be labeled on free sulfhydryl groups using maleimide reagents such as BODIPYmaleimide.…”