Infrared spectra of proteins

Baumruk V. Pancoska P. Keiderling TA. Prediction of secondary structure using statistical analysis of electronic and vibrational circular dichroism and Fourier transform infrared spectra of proteins in H2O. J Mol Biol 1996 259 774-791. 

Infrared spectra of proteins may be obtained either in the solid state (KBr pellets, crystals, or by diffuse reflectance techniques (Yang et al., 1985)) or in solution (D2O solutions, ATR techniques, as in the case of protein adsorption studies (Gendreau et al., 1982 Sec. 6.4). Raman spectra of proteins are usually obtained in solution. 

A method based on factor analysis followed by correlation of the factor loadings with structural composition has recently been proposed. This technique involves constructing a calibration set from infrared spectra of proteins whose secondary structure has been determined by X-ray. Factor analysis creates series of abstract spectra, which are combined to generate the original spectrum (Lee et al., 1990). This procedure was employed to estimate the secondary structures of membrane proteins (Lee et al., 1991). 

Due to the significant difference in the mid-infrared spectra of proteins and polysaccharides, it is possible to study simultaneously and independently their behaviours in mixtures.6 In this study ATR-FTIR was used to monitor starch gelatinisation and the evolution of the water content dependent hydrogen bonding in the gluten fraction of starch/gluten mixtures as a function of heating. 

The infrared spectra of proteins exhibit absorption bands associated with their characteristic amide group, the structural unit common to ail molecules of this type (shown in Figure 6.2a). An isolated planar amide group gives rise to five in-plane modes and one out-of-plane normal mode. The in-plane modes are due to C=0 stretching, C— N... 

The solvent environment in which the infrared spectra of proteins and peptides are recorded affects the secondary structures observed for these molecules. For example, the solvent trifluoroethanol (TFE) is more polar than water and allows different conformations to form. The FT-IR spectrum of the CiT4 peptide in a 20% TFE/80% solvent mixture was recorded and is shown in Figure 6.2f. The frequency and the fractional area of each component of the amide I... 

Very few of the infrared studies of proteins have been carried out on aqueous solutions of the proteins. Except for the work of Koenig and Tabb (1), the few aqueous IR studies have been on single proteins. Correspondingly, most of the assignments of the backbone vibrations (the so-called Amide I, II, III, etc. vibrations) have been based on either Raman spectra of aqueous solutions (2) or on infrared spectra of proteins in the solid state (3). Where infrared solution spectra have been obtained, it has mostly been on D2O solutions (4) - not H2O solutions. Since these Amide I, II, III, etc. vibrations involve motion of the protein backbone, they are sensitive to the secondary structure of the protein and thus valid assignments are necessary in order to use infrared spectroscopy for determining the conformations of proteins. 

In the past, the assignments of the protein backbone vibrations to secondary structures were often made from the spectrum of one compound and no attempts were made to support the assignments by changing the structure of the proteins. In fact structures were often determined from the frequencies and contours of one band. Only recently (4,9,10,11), have resolution enhancement or deconvolution (12) techniques being applied to the infrared spectra of proteins. While these deconvolution techniques appear to be essential for a valid interpretation of protein spectra, it is also necessary to use more than one infrared band and to substantiate assignments. 

Differences were observed between the infrared spectra of proteins from bagged blood and unmodified blood taken directly from a live animal. For example, (a) more of the 1365-cm component adsorbed from the live blood, and (b) another unknown... 

Liu, Y., Cho, R.-K., Sakurai, K., Mima, T., and Ozaki, Y, Studies on specira/structure correlations in near-infrared spectra of proteins and polypeptides. Part 1 A marker band for hydrogen bonds, Appl. Spectrosc., 48(10), 1249-1253, 1994. 

K. Murayama, B. Czamik-Matusewicz, Y. Wu, R. Tsenkova, Y. Ozaki. Comparison between conventional spectral analysis methods, chemometrics, and two-dimensional correlation spectroscopy in the analysis of near-infrared spectra of protein. Appl Spectrosc 54 978, 2000. 

Correlation Spectroscopy in the analysis of near-infrared spectra of protein. Appl. Spectmsc 54(7) 978, 2000. 

The infrared spectra of proteins and polypeptides comprise essentially four strong vibrational modes associated with the peptide link. These are the Amide A band near 3300 cm (vn-h)> the Amide I band near 1650 cm (vc=o), the Amide II band near 1550 cm " and the Amide III band near 1250 cm The latter two modes are both associated with combined stretch-... 

Infrared spectra of fats and oils are similar regardless of their composition. The principal absorption seen is the carbonyl stretching peak which is virtually identical for all triglyceride oils. The most common appHcation of infrared spectroscopy is the determination of trans fatty acids occurring in a partially hydrogenated fat (58,59). Absorption at 965 - 975 cm is unique to the trans functionaHty. Near infrared spectroscopy has been utilized for simultaneous quantitation of fat, protein, and moisture in grain samples (60). The technique has also been reported to be useful for instmmental determination of iodine value (61). 

W.R. Hruschka and K. Norris, Least squares curve fitting of near-infrared spectra predicts protein and moisture content in ground wheat, Appl. Spectrosc., 36, 261-265 (1982). 

Tabb, D. L., Koenig, J. L. Infrared Spectra of Globular Proteins in Aqueous Solution, in Analytical Applications of FT-IR to Molecular and Biological Systems, Durig, J. R. (Ed.) D. Reidel 1980, p. 241... 

The usefulness of infrared spectroscopy of proteins and membranes is increased when spectra of dry films are compared with those taken in deuterium oxide. Exchange of protons for deuterons can affect both the amide I and amide II bands. For randomly coiled proteins in D20 the amide I band is shifted down by about 10 cm."1 but for many proteins D20 does not affect the frequency of the carbonyl stretch of either the ft structure or the a-helix. In addition, upon complete exchange the amide... 

The observation by Maddy and Malcolm (53) that the amide I band of bovine erythrocyte ghosts in D20 is not shifted is remarkable because it implies that all of the membrane protein is either deeply buried in an environment of hydrophobic lipids or exists in a tightly folded a-helical conformation. We have examined extensively the infrared spectra of bovine erythrocyte ghosts, both as dry films and as intact ghosts in D20 and H20 (73). The results for dry films essentially agree with those of other workers and show no evidence of f3 structure. Little change occurs in water. In D20, however, we consistently obtained a shift in the amide I band and a considerable decrease in absorption of the amide II band. 

Figure 6. Infrared spectra of dried films of (a) butyl alcohol-extracted membrane protein and (b) beef erythrocyte membranes, taken on CaF2 plates. No shoulder characteristic of the /3 conformation occurs at 1630 cmr1...

Figure 8. Infrared spectra of erythrocyte membranes (a) in D2O-0.1 M NaCl for 1.5 hours and (b) dry. The amide I band of the wet material shows a shoulder at 1650 cm. 1, which may arise from a-helical protein...

Interpretation of Infrared Spectra in Terms of Protein Conformation. With respect to ft structure the interpretation of infrared spectra of wet and dry membranes is straightforward. There is no evidence for P conformation in any of the systems examined. Since the / structure can be produced by denaturation there appear to be no constraints... 

Hruschka, W.R. and Norris, K., Least Squares Curve Fitting of Near-Infrared Spectra Predicts Protein and Moisture Content in Ground Wheat Appl. Spectrosc. 1982, 36, 261-265. 

Numerous skin sites are possible for noninvasive near-infrared measurements.40 Examples proposed in the literature include the inner lip mucosa,26 finger,41 forearm, and tongue. In all cases, the principal spectral features of the noninvasive spectra correspond to the extent of scattering superimposed on absorption bands that originate from water, protein, and fatty tissue within the skin matrix.44,45 To a first approximation, noninvasive near-infrared spectra of skin can be fitted to a Beer-Lambert function that incorporates the additive features of absorption due to water, fat, (3-sheet protein, and type III collagen, with additional terms for tissue scattering (offset) and small temperature variations (a slope term). This function takes the following form ... 

Jackson, M.,Choo,L. P., Watson, P. H., Halliday, W. C. andMantsch,H. H. (1995) Beware of connective tissue proteins assignment and implications of collagen absorptions in infrared spectra of human tissues. Biochim. Biophys. Acta 1270, 1-6. 

In Fig. 12.2 ATR infrared spectra of starch (a complex carbohydrate) (A) soy protein (B), canola oil (mostly triglycerides) (C), and a 1 1 1 mathematical mixture of the three (D) are presented. The regions containing diagnostic bands appropriate for infrared imaging are identified with dashed boxes. Note how easily all three components may be identified in the mixture spectrum (D). 

Fourier Transform Infrared Spectroscopy (FTIR) Studies. Infrared spectra of hairless mouse stratum corneum, lipid extract and protein residue are illustrated in Figures 3 and 4 for the 4000 to 2600 cm-2 and 1800 to 1360 cm-2 regions, respectively. 

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