The current theory of pulmonary surfactant function requires that very low surface tension be achieved and maintained in the alveolar surface film during compression (expiration). To effect this condition, it has been hypothesized that the unsaturated and/or fluid components of surfactant are selectively excluded or "squeezed out" from mixed monolayers containing both saturated and unsaturated phospholipids, leaving a surface film of essentially pure 1,2-dipalmitoylphosphatidylcholine (DPPC). External reflection Fourier transform infrared (FT-IR) spectroscopy has been employed to quantitatively test this hypothesis. Mixed monolayer films of acyl chain-perdeuterated 1,2-dipalmitoylphosphatidylcholine (DPPC-d62) with 1,2-dioleoylphosphatidylglycerol (DOPG), 1-palmitoyl, 2-oleoylPG (POPG), 1,2-dipalmitoylPG (DPPG) were examined in situ at the air/water interface as a function of surface pressure. The relative intensities of CD2 (CH2) stretching vibrations of the deuterated (proteated) components permitted quantitative determination of the relative concentrations of each in the film. For 7:1 (mol:mol) mixtures of DPPC-d62/DOPG, progressive, selective squeeze out of up to about 90% of the PG component is observed over a range of surface pressures from about 51 to 68 mN/m. The extent of maximal PG squeeze out was reduced to 61% for a 7:1 (mol:mol) mixture of DPPC-d62/POPG. This phenomenon, which is at least partially reversible, appears to require relatively high rates of film compression. Squeeze out was reduced (< 20%) for 7:1 (mol:mol) mixtures of DPPC-d62/DPPG or for 7:3 mixtures of DPPC-d62/POPG. Squeeze out requires that the lipid mixture achieve surface pressures greater than about 50-60 mN/m along with unsaturation (or at least conformational disorder) in the acyl chains of the non-DPPC component.(ABSTRACT TRUNCATED AT 250 WORDS)

The major goal of this project was to use FT-IR spectroscopy to monitor the effects of chain length and temperature on small, helix-forming peptides of the general form, Ac-W(EAAAR) n A-NH 2 , where n ) 1, 3, 5, and 7, in aqueous solutions. FT-IR spectra were collected in D 2 O as a function of temperature in the range of -4 to 95°C. The spectral range of interest, 1500-1725 cm -1 , contains the amide-I′ band of the fully-exchanged H f D peptide bond. Even in these simple peptides, the amide-I′ region of the IR spectra is complex and congested, composed of features derived from the conformation of the peptide backbone and from the contributions of amino acid side chains. Unambiguous resolution of peak positions and intensities is thus extremely difficult, particularly when assessing subtle differences between two data sets. Two-dimensional correlation analysis (Noda, I. J. Am. Chem. Soc. 1989, 111, 8116. Noda, I. Applied Spectrosc. 1990 was used to guide and verify the results of the peak-fitting procedure and thereby facilitate physical interpretation of the temperature-dependent spectra. The results of the two-dimensional analysis and fitting procedure show that the spectral bands, particularly those of the amide-I′ band, exhibit significant frequency shifts and bandwidth and intensity changes as a function of temperature and chain length. For the amide-I′ modes arising from the helical and random conformations of the peptide bond, the normalization of peak intensities to units of molar absorptivity is discussed in terms of different models. Two different molar absorptivity calculations are presented, the first using the length-dependence of R-helical frequencies as predicted by perturbation theory, and the second assuming a more rigorous two-state transition. The results from each are discussed in terms of the effects of chain length on R-helix stabilization and in terms of a mechanism of helix unfolding.

Centrin is a low molecular mass (20 kDa) protein that belongs to the EF-hand superfamily of calcium-binding proteins. Local and overall changes were investigated for interactions between cations and Chlamydomonas centrin using Fourier transform infrared (FT-IR) and circular dichroic (CD) spectroscopies. FT-IR spectral features studied included the amide I' band and the side-chain absorbances for aspartate residues located almost exclusively at the calcium-binding sites in the spectral region of 1700-1500 cm(-1). The amide I' band is exquisitely sensitive to changes in protein secondary structure and is observed to shift from 1626.5 to 1642.7 cm(-1) in the presence and absence of calcium. These spectral bands are complex and were further studied using two-dimensional Fourier transform infrared (2D-FT-IR) correlation along with curve-fitting routines. Using these methods the secondary structure contributions were determined for holocentrin and apocentrin. The alpha-helical content in centrin was determined to be 60%-53% in the presence and absence of cations, respectively. Furthermore, the beta-strand content was determined to be 12%-36%, while the random coil component remained almost constant at 7%-13.5% in the presence and absence of cations, respectively. Changes in the side-chain band are mostly due to the monodentate coordination of aspartate to the cation. A shift of approximately 4 cm(-1) (for the COO- antisymmetric stretch in Asp) from 1565 to 1569 cm(-1) is observed for apocentrin and holocentrin, respectively. Thermal dependence revealed reversible conformational transition temperatures for apocentrin at 37 degrees C and holocentrin at 45 degrees C, suggesting greater stability for holocentrin.