Protein secondary structure content in solution, films and tissues: redundancy and complementarity of the information content in circular dichroism, transmission and ATR FTIR spectra.

The paper presents a simple and robust method to determine protein secondary structure from circular dichroism, transmission and attenuated total reflection (ATR) Fourier transform infrared spectra. It is found that the different spectroscopic methods bring valuable but roughly identical information on the secondary structure of proteins. ATR and transmission FTIR spectra display distinct differences, yet the secondary structure can be predicted from their spectra with roughly the same success. It is also found that one wavenumber or wavelength includes the large majority of the information correlated with secondary structure content and no more than 3 significant independent wavenumbers/wavelengths could be found for any of the spectroscopic data. This finding indicates that more complex linear combinations of the absorbance or ellipticities will not further improve secondary structure predictions. Furthermore, the information content in CD, transmission and ATR FTIR spectra is largely redundant. If combining CD and FTIR results in some improvement of structure prediction quality, the improvement is too modest to prompt spectroscopists to collect different spectroscopic data for structure prediction purposes. On the other hand, the data collected show that the quality of the FTIR spectrometers is such that biosensors or imaging methods sampling from 10(-9) to 10(-15) g yield spectra of sufficient quality to analyze protein secondary structure. These new techniques open the way to a new area of research, both in protein conformational response to ligand and imaging at sub-cellular scales.

Surface functionalization of germanium ATR devices for use in FTIR-biosensors.

Biosensors based on intrinsic detection methods have attracted growing interest. The use of Fourier transform infra-red (FTIR) spectroscopy with the attenuated internal total reflection (ATR) mode, in the biodetection context, requires appropriate surface functionalization of the ATR optical element. Here, we report the direct grafting of a thin organic layer (about 20 A depth) on the surface of a germanium crystal. This covering, constructed with novel amphiphilic molecules 2b (namely, 2,5,8,11,14,17,20-heptaoxadocosan-22-yl-3-(triethoxysilyl) propylcarbamate), is stable for several hours under phosphate buffered saline (PBS) flux and features protein-repulsive properties. Photografting of molecule 5 (namely, O-succinimidyl 4-(p-azidophenyl)butanoate) affords the activated ATR element, ready for the covalent fixation of receptors, penicillin recognizing proteins BlaR-CTD for instance. The different steps of the previous construction have been monitored by water contact angle (theta(w)) measurements, spectroscopic ellipsometry (covering depth), X-ray photoelectron spectroscopy (XPS) by using a fluorinated tag for the control of surface reactivity, and FTIR-ATR spectroscopy for the structural analysis of grafted molecules. Indeed, contrarily to silicon device, germanium device offers a broad spectral window (1000-4000 cm(-1)) and thus amide I and II absorption bands can be recorded. This work lays the foundations for the construction of novel FTIR biosensors.