Lipid/protein monolayers at the air/water interface (Langmuir films) are widely used to mimic biological interfaces such as half a lipid bilayer and the air/alveolar lining of the mammalian lung, tte advantages of monolayers as models for these interfaces arise primarily from the ease with which experimental variables such as lateral pressure, surface area, and domain structure may be controlled and compared with those ofbulk phases or with films prepared on solid substrates.

Until some 20 years ago, although a variety of physical techniques had been used to study monolayer thermodynamics and rheology, only limited information about the molecular structure and interactions of monolayer constituents could be acquired. At that time, Dluhy et al. (Dluhy 1986; Dluhy and Cornell 1985; Dluhy and Mendelsohn 1988; Mitchell and Dluhy 1988) demonstrated the feasibility of acquiring IR spectra from Langmuir films of fatty acids and phospholipids in situ at the air/water interface, tte technique used was external IR reflection-absorption spectroscopy (IRRAS), in which an IR beam impinges on the aqueous surface and is partially reflected to a detector, tte reflected radiation contains the spectrum of the monolayer constituents. A variety of studies of lipid chain conformation and phase behavior ensued, tte second generation of IRRAS applications involved determination of peptide or protein secondary structure in Langmuir films (Cornut et al. 1996; Flach et al. 1994; Pastrana-Rios et al. 1994). Technically, these experiments required the development of methods for eliminating interference from water vapor absorption (Buffeteau et al. 2000; Flach et al. 1994).

tte basic idea underlying IRRAS is straightforward. When IR radiation illuminates an aqueous film, a small fraction of the incident light is reflected, tte reflected intensity depends on the experimental geometry (angle of incidence and state of polarization of the radiation), the optical constants (real and imaginary parts of the refractive indices) of the film and subphase, and the orientation of the transition moments, tte sample vibrational modes (i.e., the IR spectrum of the film constituents) appear in the spectrum of reflected light. IRRAS spectra are presented as plots of reflectance-absorbance (RA) versus wavenumber (per centimeter). RA is defined as -\og10(R/Ro), where R is the reflectivity of the film covered surface and R0 is the reflectivity of the subphase. tte subphase is generally D20 for proteins, since the bending mode ofH20 near 1,640 cm-1 overlaps the conformation-sensitive amide I mode of proteins (l,610-l,690 cm_1). In normal transmission IR spectroscopy, absorbance values are positive numbers. In contrast, RA's may take on positive or

Springer Series in Biophysics J.L.R. Arrondo and A. Alonso Advanced Techniques in Biophysics © Springer-Verlag Berlin Heidelberg 2006

negative values depending on the experimental parameters. Previous publications provide more details regarding early advances in IRRAS (Mendelsohn et al. 1995; Mendelsohn and Flach 2002a, b).

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