Amide I region

Although the ROA spectra of typical /1-sheet proteins share some of the features observed in /3-sheet poly-L-lysine, there are also some differences, especially in the amide I region. This is because the /1-sheet in proteins tends to be twisted and irregular, whereas that in polypeptides tends to be extended, multistranded and relatively flat. 

In the amide I region there is a distinctive negative/positive couplet from 1630 to 1660 cm characteristic of a-helix, apparently in either hydrated or nonhydrated form. There are also a number of weaker positive bands in the backbone skeletal stretch region from 950 to 1000 cm 1. 

As an example, results on the adsorption of human serum albumin (HSA) at a hydrophobic polypropylene (PP) film, which was modified by PEI/PAC multilayers are shown in Fig. 10 [111]. Based on ATR-FTIR spectra in the amide I region of the adsorbed protein layer the medium kinetic course... 

Amide III VCD from aqueous solution was published in 1987 [31], and normal coordinate analyses of simple peptides and a number of isotopomers were carried out to define the exact nature of the amide III vibration [32]. Recently, we have reported a detailed comparison of computational and experimental VCD results in the amide I region [33]. Keiderling has pushed the frontiers toward collecting VCD data on a number of proteins, and interpreting the data, via factor analysis, in terms of percentages of the common secondary structures [34,35]. A number of excellent reviews, summarizing the progress in peptide VCD in the 1985-1991 time span, have appeared [36,37],... 

The development of VCD as a peptide conformational probe proceeded in a manner similar to the one followed earlier by researchers in CD spectroscopy [38], Homo-polyamino acids, such as poly-L-Tyr or poly-L-Lys, for which the secondary structure is well known and can be varied as a function of solvent acidity, were studied via VCD, and distinct results for the established conformations were observed. These results are summarized in Figures 4 and 5. In poly-L-Tyr [41], for example, the a-heli-cal conformation exhibits a distinct, sharp and near-conservative positive / negative couplet in the amide I region. In the context of this discussion, "conservative couplet" implies equal positive and negative VCD intensities, and a description of positive / negative couplet always implies low to high wavenumber. The a-helical conformation is assumed by poly-L-Tyr in acidified DMSO solution (80% 20% by volume of DMSO... 

One of the first VCD studies we undertook in 1988 was work on the tripeptide L-Ala-L-Ala-L-Ala [23], This molecule, in neutral aqueous (D O) solution, exhibits a distinct, near-conservative VCD spectrum in the amide I region, shown in Figure 7. The infrared absorption shows two overlapping peaks in the amide I region, at 1650 and 1675 cm 1. Raman depolarization ratios indicated that the high frequency component is the symmetric stretching combination. 

Fig. 6. IRRAS spectra of the lipid carbonyl and peptide amide I region (1550-1800 cm-1) for a mixed film of DPPC with 5mol% KL4 on a D20 subphase. Spectra were acquired using s-polarization, and surface pressure values are noted from top to bottom during compression (c) and expansion (e) of the film. The angle of incidence was 50°. Taken from Ref. [66] with permission from American Chemical Society.

A few scanning dispersive VCD instruments are still in use for biological applications in the mid-IR region [46,47]. In 2009, a newly designed and optimized dispersive VCD instrument was reported [47]. A collection of spectra for peptides and proteins having different dominant secondary structures (alpha-helix, beta-sheet, and random coil) measured with this new instrument showed substantially improved signal-to-noise (S/N) ratios as compared with the earlier version. The instrument provides protein VCD spectra for the amide I region that are of comparable or better quality than those obtained with a standard commercial FTIR-VCD spectrometer [47]. 

FSD spectra are frequently curve-fit to obtain an estimate of the secondary structure content of the protein being examined. This is justifiable because, in theory, Fourier self-deconvolution should not affect the relative areas of component bands. In practice however, it was found that this assumption is not valid. The relative areas of bands at the edges of the amide I region are increased by FSD. Therefore the following procedure was used for structural analysis. 

Figure 1 Comparison of infrared spectra of a-chymotrypsin in aqueous solution and dried solid state. The insert shows the second derivatives in the amide I region for the spectra in the main panel. (From Ref. 11.)...

Experimental results have shown that the hydrodynamic conformation of AFGP corresponds to an extended structure (Ahmed et al., 1975). Raman spectroscopy on this system (Tomimatsu et al., 1976) revealed that the solution spectra in the amide I region is dominated by extended conformations, consistent with the earlier results (DeVries et al., 1970), where circular dichroism also showed no extensive regions of helices. Such an open structure in turn leads to open exposure of the disaccharide groups. 

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). 

Fig. 10.5 Differential scanning calorimetry profile (a) and Amide I region in ATR-FTIR spectra (b) of cyt c in ILs (Reproduced from Ref. [7], with kind permission of The Royal Society of Chemistry)...

The fibers that are formed starting from several proteins show a characteristic infrared spectrum in the amide I region that is typical for a p-sheet structure. Structural details at the atomic level have been obtained for an amyloid forming peptide from yeast prion Sup35... 

Figure 6.2g shows the deconvolved and curve-flttec amide I region of a small synlhetic peptide Assign each of the component bands to pos sibl is secondary structures. " . 

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