Attenuated Total Reflection Infrared Spectroscopy: An Efficient Technique to Quantitatively Determine the Orientation and Conformation of Proteins in Single Silk Fibers

Abstract

Polarized attenuated total reflection (ATR) infrared spectroscopy is an efficient technique to determine the orientation and conformation of a large variety of samples, but it is more difficult to apply to very small specimens such as silk fibers. The Golden Gate single-reflection ATR accessory that uses diamond as an ATR element and a focalized beam turns out to be highly efficient to study quantitatively the orientation and conformation of a single silk fibroin filament of the silkworm Bombyx mori that is about 10 μm in diameter. For orientation measurements, rotating the sample instead of the electric field greatly simplifies the theoretical analysis and keeps the penetration depth of the infrared radiation constant. A sample holder that can be fitted on the ATR accessory has thus been developed to allow accurate rotation of the sample and to obtain spectra with a low, non-damaging, and reproducible pressure on the fiber. To validate the method, spectra have been recorded as a function of the angle θ between the fiber axis and the polarization of the incident radiation. The data have been fitted following the cosine square dependency of the absorbance with respect to the angle θ. The procedure has been applied to the spectral components of the amide I bands, as determined from spectral decomposition. Multiple angle measurements turn out to be quite useful to correct systematic angle errors and validate the accuracy of the curve-fitting parameters of the band decomposition. By using the calculated dichroic ratio, a parameter 〈P₂〉 of —0.46 ± 0.01 has been calculated for the antiparallel β-sheets and −0.04 ± 0.02 for the remaining structures. From the orientation-insensitive spectrum A₀, the amount of β-sheets has been estimated to 49 ± 3%. The results obtained from only two measurements with the electric field of the incident radiation parallel and perpendicular to the fiber axis has demonstrated that ATR spectroscopy can be used routinely in quantitative studies of the molecular orientation and conformation of macromolecules.