Location of structural transitions in an isotopically labeled lung surfactant SP-B peptide by IRRAS

Abstract

Pulmonary surfactant, a lipid/protein complex that lines the air/water interface in the mammalian lung, functions to reduce the work of breathing. Surfactant protein B (SP-B) is a small, hydrophobic protein that is an essential component of this mixture. Structure-function relationships of SP-B are currently under investigation as the protein and its peptide analogs are being incorporated into surfactant replacement therapies. Knowledge of the structure of SP-B and its related peptides in bulk and monolayer phases will facilitate the design of later generation therapeutic agents. Prior infrared reflection-absorption spectroscopic studies reported notable, reversible surface pressure-induced antiparallel beta-sheet formation in a synthetic peptide derived from human SP-B, residues 9-36 (SP-B(9-36)). In the current work, infrared reflection-absorption spectroscopy is applied in conjunction with isotopic labeling to detect the site and pressure dependence of the conformational change. SP-B(9-36), synthesized with (13)C=O-labeled Ala residues in positions 26, 28, 30, and 32, shifted the beta-sheet marker band to approximately 1600 cm(-1) and thus immediately identified this structural element within the labeled region. Surface pressure-induced alterations in the relative intensities of Amide I band constituents are interpreted using a semiempirical transition dipole coupling model. In addition, electron micrographs reveal the formation of tubular myelin structures from in vitro preparations using SP-B(9-36) in place of porcine SP-B indicating that the peptide has the potential to mimic this property of the native protein.

FIGURE 1

(A) Sequence of human SP-B and (B) the SP-B_9–36 peptide with ¹³C=O labeled Ala residues denoted with (^*). (C) Secondary structure predictions and hydrophobicity index for human SP-B calculated using PredictProgram (see text for references). The propensities for the 9–36 peptide are highlighted within the dashed line region.

FIGURE 2

Bulk phase IR spectra of the Amide I and II region for the labeled and unlabeled SP-B_9–36 peptides in methanol at ∼2 mg/mL.

FIGURE 3

IRRAS spectra (1750–1540 cm⁻¹ region) of native, porcine SP-B, and the labeled and unlabeled SP-B_9–36 peptide monolayers acquired using s-polarization at surface pressure of ∼27 mN/m on a D₂O-buffered subphase.

FIGURE 4

IRRAS spectra of the Amide I region acquired for ^*SP-B_9–36 on a D₂O subphase. (A) Spectra are acquired using s-polarization and surface pressure values are noted from top to bottom during compression and subsequent expansion of the monolayer. The inset displays the pressure dependence of the relative area of the curve fit 1597 and 1640 cm⁻¹ Amide I band components during intermittent compression (•) and expansion (○) of the film. (B) Spectra acquired using p-polarized radiation during the compression of an ^*SP-B_9–36 peptide monolayer.

FIGURE 5

Electron micrographs of (A) tubular myelin isolated from rat lungs, bar = 500 nm; (B) an in vitro preparation of TM using native, porcine SP-B, bar = 500 nm; and (C) TM prepared in vitro using SP-B_9–36 in place of native SP-B, bar = 250 nm.

FIGURE 6

Calculated IR spectra of the Amide I region using a transition dipole coupling method as described by Brauner et al. (2000). (A) Simulations of three peptides, 12 residues in each strand, arranged in an antiparallel β-sheet geometry: (—) all unlabeled residues, (– –) alternating ¹³C=O/¹²C=O, and (– · –) all ¹³C=O labeled residues. (B) Experimental spectrum of ^*SP-B_9–36 acquired at a pressure of 4 mN/m overlaid with an inverted simulated spectrum of two identical peptides with their C-termini in an antiparallel β-sheet geometry. Each peptide consists of 14 residues in an α-helical geometry coupled to an antiparallel β-sheet strand containing seven residues with ¹³C=O labels as noted (•) in inset. (C) Experimental spectrum acquired at a pressure of 29 mN/m and simulated spectrum similar to B, except that the β-strand portion of each peptide consists of 11 residues, with the labeling differing as noted in the inset. The β-sheet components in the simulated spectra in B and C were multiplied by 1.5 before being added to the helical component.