Fourier transform infrared studies of secondary structure and orientation of pulmonary surfactant SP-C and its effect on the dynamic surface properties of phospholipids

Abstract

SP-C, a highly hydrophobic, 3.7-kDa protein constituent of lung surfactant, has been isolated from bovine lung lavage, purified, and reconstituted into binary lipid mixtures of 1,2-dipalmitoyl-phosphatidylcholine (DPPC) and 1,2-dipalmitoylphosphatidylglycerol (DPPG). Fourier transform infrared (FT-IR) spectroscopy has been applied to examine SP-C secondary structure, the average orientation of alpha-helical segments relative to the bilayer normal in membrane films, and the effect of protein on the thermotropic properties of the phospholipid acyl chains. In addition, dynamic surface measurements were made on phospholipid films at the A/W interface in the presence and absence of SP-C. SP-C (0.5 mol %) was found to possess about 60% alpha-helical secondary structure in lipid vesicles. Higher levels (1.5 mol %) of SP-C resulted in a slight increase of beta-forms, possibly resulting from protein aggregation. The helical segments exhibited an average angle of orientation of about 24 degrees with respect to the bilayer normal, suggesting a trans-bilayer orientation of the peptide. The observation that 70% of the peptide bond hydrogens are hard to exchange in D2O further reflects the hydrophobic nature of the molecule. SP-C produced little effect on the thermotropic properties of the binary lipid mixture, as measured from acyl chain C-H and C-D stretching frequencies. However, the presence of 1 mol % protein markedly reduced the viscance and increased the elasticity of surface films suggesting a mechanism by which SP-C facilitates the spreading of phospholipids on an aqueous surface. The possible physiological consequences of these observations are discussed.

Phosphorylation-induced Conformational Changes of Cystic Fibrosis Transmembrane Conductance Regulator Monitored by Attenuated Total Reflection-Fourier Transform IR Spectroscopy and Fluorescence Spectroscopy

“…Presently, such a high exchange rate has been shown only for a few membrane proteins like, for example, 90 -95% of exchange for the lactose permease (39), and 81% for the human erythrocyte glucose exchanger (40) after 1 h of deuteration. These proteins present an exchange behavior clearly distinct from that encountered for most of the membrane proteins where almost no exchange occurs in the membrane domain (41)(42)(43)(44). These fast exchanging proteins share the common function of transporting relatively large hydrophilic molecules, and consequently it is expected that they should possess a large hydrophilic pore.…”

Molecular Structures and Interactions of Pulmonary Surfactant Components

“…The a-helix of SP-C seems to be a very rigid and stable structure. It was observed by FTIR spectroscopy that 60% of the polypepide chain amide protons of native or depalmitoylated porcine SP-C (Vandenbussche et al, 1992b) and 70% of calf SP-C (Pastrana et al, 1991) did not exchange with 'H'O. This was confirmed by NMR spectroscopy of SP-C in all-deuterated solvents which showed that the amide protons from positions 10-32 exchange with a rate <8.3X10~-4/min (Johansson et al, 1994 b).…”

The Pulmonary Surfactant System: Biochemical and Clinical Aspects

“…The protein is extremely hydrophobic, and is characterized by a high content of valine residues. Two thirds of the protein consists of a continuous hydrophobic stretch, and the secondary structure of this part of the protein is a regular ␣-helix [154,244,286] which is able to span a DPPC bilayer [216]. It has been shown that the long axis of the ␣-helix is oriented parallel to Table 3.…”