Amide vibration

Chi Z H, Chen X G, Holtz J S W and Asher S A 1998 UV resonance Raman-selective amide vibrational enhancement quantitative methodology for determining protein secondary structure Biochemistry 27 2854-64... 

Characterization of amide vibrational modes as seen in IR and Raman spectra has developed from a series of theoretical analyses of empirical data. The designation of amide A, B, I, II, etc., modes stem from several early studies of the (V-methyl acetamide (NMA) molecule vibrational spectra which continues to be a target of theoretical analysis. 15 27,34 162 166,2391 Experimental frequencies were originally fitted to a valence force field using standard vibrational analysis techniques and subsequently were compared to ab initio quantum mechanical force field results. 

Table 4-6 lists observed frequencies and band assignments of structure-sensitive amide vibrations. Here, we discuss only amide I and III bands for which abundant data are available. The general trends shown in the table below were found by correlating x-ray structural data with Raman frequencies. 

Table 4-6 Band Assignments of Amide Vibrations in /V-Methylacetamide (cm 1)...

Similar procedures might turn out to be necessary in the case of 2D vibrational spectroscopy. The spectroscopy demonstrated so far essentially reflects the first step of the procedure in NMR spectroscopy (COSY). So far, we have investigated only the amide I subspace. However, all amide vibrations (N-H, amide II, etc.) might turn out to be equally important in revealing the required information, each of them giving different and hopefully complementary pieces of information. Couplings between different amide subspaces need to be explored. Incoherent population transfer out of the amide I transition appears to be very efficient (Ti = 1.2 ps), and the mechanism and the course of this transfer is completely unknown. 

UV resonance Raman spectroscopy (UVRR), Sec. 6.1, has been used to determine the secondary structure of proteins. The strong conformational frequency and cross section dependence of the amide bands indicate that they are sensitive monitors of protein secondary structure. Excitation of the amide bands below 210 nm makes it possible to selectively study the secondary structure, while excitation between 210 and 240 nm selectively enhances aromatic amino acid bands (investigation of tyrosine and tryptophan environments) (Song and Asher, 1989 Wang et al., 1989, Su et al., 1991). Quantitative analysis of the UVRR spectra of a range of proteins showed a linear relation between the non-helical content and a newly characterized amide vibration referred to as amide S, which is found at 1385 cm (Wang et al., 1991). 

Detailed analyses of the vibrational spectra of raacromolecules, however, have provided a deeper understanding of structure and interactions in these systems (Krimm, 1960). An important advance in this direction for proteins came with the determination of the normal modes of vibration of the peptide group in A -methylacetamide (Miyazawa et al., 1958), and the characterization of several specific amide vibrations in polypeptide systems (Miyazawa, 1962, 1967). Extensive use has been made of spectra-structure correlations based on some of these amide modes, including attempts to determine secondary structure composition in proteins (see, for example, Pezolet et al., 1976 Lippert et al., 1976 Williams and Dunker, 1981 Williams, 1983). 

An example for the determination of the kinetics of a solid-phase reaction via ATR-spectroscopy is shown in Figure 16.6. The cyclo-addition reaction leading to substituted isoxazolidines is initialized, and resin samples are taken out of the reaction mixture after certain time intervals [31]. ATR spectra were recorded to determine the relative intensities of the carbonyl vibration (v (C=0) = 1715 cm ).The spectra were normalized using the amide vibration (v(C(0)NFI2) = 1682 cm-1). 

In addition, the infrared contributions of the side chains of the amino acids which constitute the protein must be considered. Amino acid side chains exhibit infrared modes that are often useful for investigating the local group in a protein. It is also important to be aware of the location of such modes as they may be confused with amide vibrations. Fortunately, these contributions have been found to be small in D2O when compared to the contributions made by the amide I band. The characteristic side-chain infrared frequencies of amino acids are summarised in Table 6.2b. 

The frequencies of the infrared peptide backbone vibrations (the so-called Amide vibrations) of proteins can be used to differentiate secondary structures (conformations) in proteins in aqueous solutions. 

A number of characteristic SERS bands originate from the amino acid side chains Trp, Tyr and Phe. The peptide backbone vibrations are not enhanced in this protein (low scattering intensity in the spectral range of 1650-1675 cm amide I). The presence of the strong SERS bands of Trp, Tyr and Phe and the absence of the amide vibrations indicate a preferential interaction of these amino acid side chains with the surface. The strong (S—S) vibration at 509 cm in the NSRS spectrum is also missing in the SERS spectrum. This indicates that the disulfide bonds do not interact directly with the surface. 

Figure 7.6 Absorption line scan across a sharp edge in a polymer photoresist using the X, = 6 pm amide vibration spectral feature. Raw absorption signal as a function of distance (open circles) and the expected profile (solid line) assuming a perfectly sharp edge convolved with a calculated point spread function.

IR studies, in this way the overlapping can be avoided since vibrational frequencies are usually shifted to lower values for proteins in D O as compared to H O solutions. The amide II band appears in the 1560-1510 cm range with strong-medium intensity. The other visible amide vibrational mode corresponds to the amide 111 band which falls in the 1300-1200 cm region. 

Figure I presents FTIR/ATR spectra of live and treated cryptococci H99 (A, B) and d plbl cells (C, D). The spectra of the live and heat treated samples are dominated by the amide I (1639 cm" ) and II (1547 cm" ) bands which are generated by the peptide bond formed between amino acid residues within a polypeptide chain or protein (26). These amide vibrations are attributed to the mannoprotein component of the capsule and cell wall of Cryptococcus (23, 27). The other prominent feature is the broad intense peak centred at -1024 cm" which is attributed to numerous v(C-0) vibrational modes from polysaccharides also present within the capsule and cell wall (28).

The large number of normal modes of biomolecules is also associated with an experimental difficulty inasmuch as individual Raman-active modes may be closely spaced so that the observed peaks include several unresolved bands. This is particularly true for those modes which originate from chemically identical building blocks of the biopolymers such as the amide vibrations of proteins. In these cases, however, the analysis of these bands provides valuable information about the structure of the ensemble of these building blocks, e.g. the secondary structure of proteins. 

Costa et al. [18] employed ATR-FTIR spectroscopy to analyze the interactions between biopolymers and P(NiPAM-co-MAA) MGs (Figure 6.10). The spectra are difficult to analyze since the characteristic PE bands overlap with the amide vibrations. Moreover, the exact band positions for the polypeptides depend on their conformation (a-helix, p-sheet, or random coil), due to vibrational interactions between the peptide groups and the... 

---