Quantitation of secondary structure in ATR infrared spectroscopy

Polarized attenuated total reflection infrared spectroscopy of aligned membranes provides essential information on the secondary structure content and orientation of the associated membrane proteins. Quantitation of the relative content of different secondary structures, however, requires allowance for geometric relations of the electric field components (E(x), E(y), E(z)) of the evanescent wave, and of the components of the infrared transition moments, in combining absorbances (A and A(perpendicular)) measured with radiation polarized parallel with and perpendicular to, respectively, the plane of incidence. This has hitherto not been done. The appropriate combination for exact evaluation of relative integrated absorbances is A + (2E(z)(2)/E(y)(2) - E(x)(2)/E(y)(2))A(perpendicular), where z is the axis of ordering that is normal to the membrane plane, and the x-axis lies in the membrane plane within the plane of incidence. This combination can take values in the range approximately from A - 0.4A(perpendicular) to A + 2.7A(perpendicular), depending on experimental conditions and the attenuated total reflection crystal used. With unpolarized radiation, this correction is not possible. Similar considerations apply to the dichroic ratios of multicomponent bands, which are also treated.