Amide Raman bands

It is a supposition that the )9-sheet structure of neurotoxin is an essential structural element for binding to the receptor. The presence of -sheet structure was found by Raman spectroscopic analysis of a sea snake neurotoxin (2). The amide I band and III band for Enhydrina schistosa toxin were at 1672 cm and 1242 cm" respectively. These wave numbers are characteristic for anti-parallel -sheet structure. The presence of -sheet structure found by Raman spectroscopic study was later confirmed by X-ray diffraction study on Laticauda semifasciata toxin b. 

Silk fibers, a basic system with a uniaxial symmetry, have also been investigated by Raman spectromicroscopy [63] that is one of the rare techniques capable of providing molecular data on such small (3-10 pm diameter) single filaments. The amide I band of the silk proteins has been particularly studied to determine the molecular orientation using the cylindrical Raman tensor approximation. In this work, it was assumed that Co Ci, C2 and the a parameter was determined from an isotropic sample using the following expression of the depolarization ratio... 

Figure 10 shows polarized spectra of two types of silks recorded by Raman spectromicroscopy the dragline silk (the lifeline) of the spider Nephila edulis and the cocoon silk of a wild silkworm Sarnia cynthia ricini. The position of the amide I band at 1,668-1,669 cm-1 for both threads is characteristic of the /i-sheet... 

In the literature Raman spectroscopy has been used to characterize protein secondary structure using reference intensity profile method (Alix et al. 1985). A set of 17 proteins was studied with this method and results of characterization of secondary structures were compared to the results obtained by x-ray crystallography methods. Deconvolution of the Raman Amide I band, 1630-1700 cm-1, was made to quantitatively analyze structures of proteins. This method was used on a reference set of 17 proteins, and the results show fairly good correlations between the two methods (Alix et al. 1985). 

Both amide I and amide III bands are seen in Raman spectra of proteins.30 Lippert et al. devised the following method for estimating the fractions of a-helix, (3 sheet, and random coil conformations in proteins.31 The amide I Raman bands are recorded at 1632 and 1660 cm 1 in DzO (amide I ). The amide III band, which is weak in DzO, is measured at 1240 cm-1 in H20. The intensities of the three bands relative to the intensity of an internal standard (the 1448 cm 1 CH2... 

The frequency of the amide I peak observed in the lens is sensitive to protein secondary structure. From its absolute position at 1672 cm-1, which is indicative for an antiparallel pleated 3-sheet structure, and the absence of lines in the 1630-1654 cm-1 region, which would be indicative of parallel (1-sheet and a-helix structures, the authors could conclude that the lens proteins are all organized in an antiparallel, pleated 3-sheet structure [3]. Schachar and Solin [4] reached the same conclusion for the protein structure by measuring the amide I band depolarization ratios of lens crystallins in excised bovine lenses. Later, the Raman-deduced protein structure findings of these two groups were confirmed by x-ray crystallography. 

Vibrational spectroscopy has been used in the past as an indicator of protein structural motifs. Most of the work utilized IR spectroscopy (see, for example, Refs. 118-128), but Raman spectroscopy has also been demonstrated to be extremely useful (129,130). Amide modes are vibrational eigenmodes localized on the peptide backbone, whose frequencies and intensities are related to the structure of the protein. The protein secondary structures must be the main factors determining the force fields and hence the spectra of the amide bands. In particular the amide I band (1600-1700 cm-1), which mainly involves the C=0-stretching motion of the peptide backbone, is ideal for infrared spectroscopy since it has an large transition dipole moment and is spectrally isolated... 

There are a number of methods based on using the spectra of proteins with secondary structures known from X-ray data [707]. An example is the method of Williams [8,116] which analyzes the amide I band of the Raman spectrum for a protein of unknown structure in terms of linear combinations of amide I bands for proteins with known X-ray structure. Significant correlations were observed between the Raman and X-ray diffraction estimates of helix, P-strand, turn, and undefined. Correlations were also observed between a-helix and disordered helix, and between parallel and antiparallel p-sheets. 

Baron and de Loz6212 have studied the interaction of several salts with NMA using IR and Raman spectroscopy. It was found that, in very concentrated solutions, even tetra-n-butylammonium bromide caused shifting of the amide carbonyl bands... 

Yu (unpublished work quoted by Yu, 1974) found that, as would be expected, there was no sign of conformational change for lysozyme, as revealed by the Raman spectrum, as the pH was decreased from 5.2 to 2.0. In particular, there was virtually no change in the amide III backbone region (1220—1300 cm ). On the other hand, there were changes in amide III frequencies and the contour of Raman bands for a-lactalbumin upon the same reduction in pH value. 

The amide V mode, similar to (Gly) I, appears as a strong IR band at 706 cm" . The essential absence of any significant dichroism in this band in well-oriented specimens of )3-(Ala)n (Itoh et al., 1968) suggests the near superposition of components having parallel and perpendicular polarization. As can be seen, this is supported very well by the results of the calculation, which additionally indicate that a weak Raman band at 698 cm" should be assigned to amide V. 

Despite our observations that amide V seems to be associated with a characteristically strong IR band near 705 cm", no such band is found for j8-(GluCa) , although a weak Raman band at 705 cm" [cf. the comparable 698-cm" band in /3-(Ala) ] does qualify as such a mode by its disappearance on N-deuteration. However, the mainly CO ob mode, which is observed at 614M cm" in (Gly) I and is not observed in j8-... 

The predictions for amide I and II modes are generally good, considering that force constants were transferred without further refinement. The large frequency difference between the 1680 (IR) and 1691 cm (Raman) bands may reflect the presence of intermolecular interactions in the crystal. In any event, we do not expect frequencies as high as these for a standard type II jS turn. Their observation and prediction are undoubtedly related to the particular structure of this molecule, which emphasizes the caution required in assuming general characteristic turn frequencies. Incidentally, the calculation predicts that the 1658-... 

In Table XXVI we also list the observed IR and Raman bands in the amide III and V regions that weaken or shift on N-deuteration, and the calculated modes containing NH ib or NH ob, respectively, that can be assigned to them. Despite the prediction for the standard turn that amide III frequencies should occur above about 1300 cm" (see Table XXI), we find two clearly N-deuteration-sensitive bands below, at 1271 and 1241 cm" , that are well accounted for by the calculation. This arises from the different forms of the normal modes for the two molecules, particularly the C C > (Leu) s contribution to the 1237-cm" mode. A similar consideration may apply to the 687-cm (IR) band, which is much higher than the highest predicted mode of the standard turn The CH2 r contribution in the latter case is absent for the tripeptide. 

Fig. 29. (a) Calculated frequencies in the amide I region for the 10 lowest energy conformations of cyclo(L-Ala—Gly-Aca) (see Table XXVII). The observed infrared (solid bar) and Raman (open bar) bands are shown on the bottom line. Numbers above the computed frequencies represent the groups involved in the vibration (Maxfield et al., 1981). (b) Calculated frequencies in the amide V region for the 10 lowest energy conformations of cyclo(i.-Ala—Gly-Aca) (see Table XXVII). The observed infrared and Raman bands occur at the same frequencies and are indicated by the shaded bars on the bottom line. Numbers above the calculated frequencies represent the groups involved in the vibration (Maxfield et al., 1981). 

The use of polarized light to generate contrast between bone components provides information on the spatial distribution of bone components and their orientation. Kazanci et al. used Raman polarized imaging to examine the distribution of mineral and matrix constituents around osteons, and showed that the POi Vi and amide I bands are highly orientation-dependent, whereas the amide III and POi V2 and V4 are less orientation-dependent [35]. Orientation effects are nicely illustrated in Figure 4.2 [36]. The POi Vj amide I ratio coincides with the orientation of the lamellae the same lamellae have different contrasts, depending on the polarization of the excitation light and the orientation of the specimen. 

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