Attenuated Total Reflection Infrared Spectroscopy: An Efficient Technique to Quantitatively Determine the Orientation and Conformation of Proteins in Single Silk Fibers MAXIME BOULET-AUDET, THIERRY LEFE ` VRE, THIERRY BUFFETEAU, and MICHEL PE ´ ZOLET* Centre de Recherche en Sciences et Inge ´nierie des Macromole ´cules, Centre de Recherche sur la Fonction, la Structure et l’Inge ´nierie des Prote ´ines, De ´partement de Chimie, Universite ´ Laval, Pavillon Alexandre-Vachon, Que ´bec, G1V 0A6, Canada (M.B.-A., T.L., M.P.); and Institut des Sciences Mole ´culaires, UMR 5255 du CNRS, Universite ´ de Bordeaux I, 33405 Talence, France (T.B.) 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 lm 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 h 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 h. 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 hP 2 i of 0.46 6 0.01 has been calculated for the antiparallel b-sheets and 0.04 6 0.02 for the remaining structures. From the orientation-insensitive spectrum A 0 , the amount of b-sheets has been estimated to 49 6 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. Index Headings: Golden Gate; Infrared spectroscopy; IR spectroscopy; Attenuated total reflection; Diamond ATR; Silkworm cocoon; Silk fiber; Silk fibroin; Molecular orientation; Secondary structure. INTRODUCTION Fourier transform infrared (FT-IR) spectroscopy is a valuable technique to characterize the molecular conformation and orientation of natural and synthetic macromolecular systems since it can provide high quality spectra of minute amounts of material. In addition, because of the wide variety of available sampling techniques, samples of different size, shape, and physical state can be investigated. Although thin films and solutions can easily be studied by transmission infrared spectroscopy, it is more difficult to obtain quantitative information about the conformation and orientation of small single fibers such as silkworm and spider silk fibers because their size is often below the diffraction limit of most infrared microscopes. Arrays of tightly packed fibers can be used for transmission experiments, but this method rarely provides high quality spectra because of spectral artifacts due to sample heterogeneities and appreciable leakage of incident radiation through the sample. 1,2 Finely cut fibers can also be studied by diffuse reflectance infrared spectroscopy or by transmission spectroscopy when dispersed in KBr pellets. 3 Unfortunately, the microstructure of the fibers is not necessarily preserved with these methods and the information on the molecular orientation is completely lost. Since molecular orientation strongly affects the mechanical properties of natural and synthetic fibers, it is important to develop techniques that allow the efficient and quantitative determination of molecular orientation in situ in single fibers. One of the most efficient techniques to study fiber microstructure by infrared spectros- copy is undoubtedly attenuated total reflectance (ATR). The main limitation of most conventional ATR accessories is that several filaments are often necessary to obtain spectra with a sufficiently high signal-to-noise ratio. This strategy has been implemented to examine the conformation of hair fibrous proteins 4,5 and textile polymers. 6–8 Three methods have been used to determine molecular orientation by ATR infrared spectroscopy. The most common one is to keep the sample fixed on the ATR element and to rotate the electric field of the incident radiation. This method is simple to implement since the sample does not have to be moved, but it requires complete knowledge of the components of the electric field in every direction at the surface of the ATR crystal. 9–11 Depending on the thickness of the sample, the ATR element, and the sample, the determination of the electric fields requires the knowledge of the optical properties of the multilayer system. 12 A second method consists of rotating the sample and ATR crystal as a whole, 13 thus ensuring a constant contact between the sample and the ATR crystal. However, it has been demonstrated that a slight change in the beam path has an important effect on the reproducibility of the measurements. 14 The third method consists of rotating the sample while keeping the beam path and polarization unchanged. The main limitation of this technique is that the contact between the sample and the ATR element can change when the sample is rotated, which strongly affects the intensity of the polarized spectra. To minimize this effect, normalization of the spectra is necessary and can be performed using an orientation-independent band. When such a band is not present, a dye molecule can be incorporated in the sample to normalize the spectra. Such a procedure has been used to determine Received 24 April 2008; accepted 9 June 2008. * Author to whom correspondence should be sent. E-mail: michel.pezolet@ chm.ulaval.ca. 956 Volume 62, Number 9, 2008 APPLIED SPECTROSCOPY 0003-7028/08/6209-0956$2.00/0 Ó 2008 Society for Applied Spectroscopy