FTIR Spectral Analysis

Infrared spectra of proteins in aqueous media exhibit nine characteristic absorption bands, which represent different vibration modes of their peptide moiety. Among all the amide vibrational modes, the most frequently used in studies of protein secondary structure is the amide I band. This vibrational mode, which lies in the 1,600-1,700 cm-1 spectral range, corresponds to the stretching mode of the peptide carbonyls. This broad infrared absorption band, which is sensitive to protein conformation, is composed of multiple component bands whose wavenumber depends on whether the peptide carbonyls are hydrogen bonded to other peptide units, or to side-chains, or to the solvent. Each component band could be assigned to a different structural element such as the a-helix, ^-sheet, or "free" peptide groups located in random hydrophobic or polar domains. Spectral correlation with the secondary structure in the amide I spectral region is well documented on model polypeptides (de Loze and Fillaux 1972; Tsuboi 1964) and on a large number of globular proteins (Byler and Susi 1986; Goormaghtigh et al. 1994; Kalnin et al. 1990; Krimm and Bandekar 1986).

Because the broad amide I band is made of overlapping component bands, resolution enhancement techniques are necessary to extract useful structural information from the infrared spectra of proteins. The main mathematical methods used to analyse these infrared spectra are self-deconvolution or second-derivative processing and curve-fitting, and are well described in the literature (Dong et al. 1990; Dousseau and Pezolet 1990; Surewicz et al. 1993).

Prior to the infrared analysis, subtraction of the water contribution must be undertaken. Even if many algorithms are now available to subtract the water contribution from the infrared spectra of proteins, this protocol should be used with caution when dealing with adsorbed proteins. Indeed, the adsorption process could induce a change in solvation of both the proteins and the solid support. As a result, the water molecules present at the interface could be involved in hydrogen bonding, which shifts the broad water contribution band and render the subtraction oftheir infrared contribution difficult (Giacomelli et al. 1999a). The main advantage of performing infrared analysis of proteins in D2O is to allow a direct comparison between the solvated state and the adsorbed state of the protein. With regard to determination of the secondary structure, a few minutes of incubation of the protein in a deuterated medium is enough to shift the amide I band from 2 to 10 cm-1. Assignments of component bands of the so-called amide I' band are reported in different works on globular proteins (Chirgadze and Brazhnikov 1974; Goormaghtigh et al. 1994). The great advantage is the shift of the disordered component band of amide I centred at 1,650 cm-1 in H2O to 1,640 cm-1 in D2O, whereas the component bands relative to ordered structures (a-helices, ^-sheets) are less affected upon deuteration (Wantyghem et al. 1990).

In our procedure, the curve fitting is performed on both the amide I' and amide II spectral regions and calculated as a linear combination of individual band components by iterative adjustments of only the heights of the components, the other parameters like profiles, widths and wavenum-bers being fixed. Then the fractional areas of the band components are assumed to represent percentages of the different types of ordered or random domains of the protein. Since FTIR spectra and mathematical decompositions are all run in the same way, the resulting difference in the spectral analyses between the adsorbed state and the state in solution reveals semi-quantitatively the structural transition upon adsorption (Baron et al. 1999; Noinville et al. 2002; Revault et al. 2005; Servagent-Noinville et al. 2000; Wantyghem et al. 1990).

Moreover, the use of deuterated media enables the researcher to follow the NH/ND exchange of the all amide protons of the protein backbone by examining the reduction of the amide II band. Since the NH/ND exchange rate slows down with the strength of hydrogen bonding involved in stereo-

regular structures, it is also possible to extract information on the protein stability.

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