Analysis of the Amide I Band

Two types of secondary structure analysis were used on the spectra collected in this study. The classic curve-fitting method (13) for analysis of the amide I band, was performed in two stages. The first step in the analysis is band narrowing, which allows visualization of component bands, using derivatization and Fourier self-deconvolution (FSD) (14). 

Table 6.2h Analysis of the amide I band of the CiT4 peptide in TFE/D2O...

The analysis of the amide I band to obtain the estimation of protein secondary structure content in terms of percentage helix, j3 strand, and reverse turn that was developed by Williams has proved very successful and has now been used by numerous workers.In this method the amide I region is analyzed as a linear combination of the spectra of the reference proteins whose structures are known. As noted above the Raman spectra of globular proteins in the crystal and in solution are almost identical, reflecting the compact nature of the macromolecules. Thus one may use the fraction of each type of secondary structure determined in the crystalline state by the X-ray diffraction studies for proteins in solution. If there are n reference proteins with the Raman spectrum of each of them represented as normalized intensity measurements at p different wave numbers, then this information is related by the following matrix equation ... 

To apply the analysis of the amide I band to proteins for which a rigorous normal mode analysis is not available, a more empirical approach has been adopted. Because the amide I band of proteins is very broad and featureless, a direct analysis of the band in terms of secondary structural elements is not possible. However, if the band is subjected to so-called resolution-enhancement data analysis, several individual bands can be extracted. Spectral deconvolution and derivative techniques are applied (the latter is a special case of the former). To avoid artifacts, spectra with very high signal/noise ratios have to be measured. Here, the advantage... 

Table 7.5 Analysis of the amide I band of lysozyme in D O. From Stuart, B., Biological Applications of Infrared Spectroscopy, ACOL Series, Wiley, Chichester, UK, 1997. University of Greenwich, and reproduced by permission of the University of Greenwich...

Secondary structure fractions were estimated by the Raman spectral analysis program of Przybycien and Bailey (1989), based on the algorithms of Williams (1983) for least-squares analysis of the Amide I band. 

Torii H, Tasumi M. Theoretical analysis of the amide I infrared bands of globular proteins. In Mantsch HH, Chapman D, eds. Infrared Spectroscopy of Biomolecules. New York Wiley-Liss, Inc., 1996 1-18. 

Fig. 10.15. Ratio of absorbance at 1513cm of deuterated proteins (Dr-) to absorbance of the amide I band (/>,) plotted against tyrosine content from amino acid analysis. Values of Dr/Di for silk, gliadin, and pepsin are based on spectra of partially deuterated specimens all the other proteins were fully or almost fully deuterated. (Bendit, 1967.)...

Fourier-transformed infrared (FTIR) is another excellent method to study protein folding. Unlike the well-known use of FTIR as a method for the identification of functional groups, in terms of protein structure this method allows the determination of secondary structure. The frequency of vibration of the amide I band of the peptide chain (1500-1600 cm M heavily depends on the structure of the protein. FTIR has the advantage of being more sensitive for the study of proteins that contain (3-sheet elements as compared to CD. Furthermore, since FTIR spectroscopy can be applied to solids also, it allows the structural analysis of aggregated protein deposits. The availability of the rapid step-scan method for FTIR is also very useful for the study of rapid folding reactions (see Vibrational Spectroscopy). 

The ability to predict a band intensity profile opens up an important additional dimension in vibrational analysis. It means that we will be able to relate subtle spectral differences to small structural changes with a greater degree of confidence. Thus, it has been possible to confirm that an observed three-component contour of the amide I band of tropomyosin is indeed expected for a coiled-coil a-helix [142]. Extensions to understanding the normal modes of proteins become possible [143]. The systematic incorporation in an SDFF of dipoles and dipole fluxes to calculate IR intensities [144] will finally bring to the vibrational analysis of polymeric molecules the completeness and flexibility needed to make it a much more powerful structural tool. 

The amide I band was chosen for detailed analysis as its position is sensitive to protein secondary structure. The band arises predominately from v(C=0) stretching of the carbonyl group within the peptide (CONH) bond. Other minor contributing factors arise from v(CN) out-of-plane, 5(CCN) and 8(NH) in-plane vibrations (1). The broad nature of the amide I band is attributed to the presence of a number of secondary structures within the sample. Derivatives were used to deconvolve spectral band widths and positions. This resolution technique, together with deconvolution and curve fitting, is particularly useful for resolving components within a broad band envelope. 

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. 

Fig. 9.28 Analysis of the CH-stretching region (3000-2800 cm ) and the amide I band around 1650 cm V (a) ER-FTIR spectrum of poly(2-ethyl-2-oxazoline) (PEOx) as grown on the triflate functionalized HUT SAM. (b) ER-FTIR spectrum of HUT SAM. (c) Subtraction result of (a)-(b). (d) Bulk spectrum of PEOx. In the spectrum to the left, a significant shift...

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. 

The particle beam LC/FT-IR spectrometry interface can also be used for peptide and protein HPLC experiments to provide another degree of structural characterization that is not possible with other detection techniques. Infrared absorption is sensitive to both specific amino acid functionalities and secondary structure. (5, 6) Secondary structure information is contained in the amide I, II, and III absorption bands which arise from delocalized vibrations of the peptide backbone. (7) The amide I band is recognized as the most structurally sensitive of the amide bands. The amide I band in proteins is intrinsically broad as it is composed of multiple underlying absorption bands due to the presence of multiple secondary structure elements. Infrared analysis provides secondary structure details for proteins, while for peptides, residual secondary structure details and amino acid functionalities can be observed. The particle beam (PB) LC/FT-IR spectrometry interface is a low temperature and pressure solvent elimination apparatus which serves to restrict the conformational motions of a protein while in flight. (8,12) The desolvated protein is deposited on an infrared transparent substrate and analyzed with the use of an FT-IR microscope. The PB LC/FT-IR spectrometric technique is an off-line method in that the spectral analysis is conducted after chromatographic analysis. It has been demonstrated that desolvated proteins retain the conformation that they possessed prior to introduction into the PB interface. (8) The ability of the particle beam to determine the conformational state of chromatographically analyzed proteins has recently been demonstrated. (9, 10) As with the ESI interface, the low flow rates required with the use of narrow- or microbore HPLC columns are compatible with the PB interface. 

In the IR spectra (table 2) of the encapsulated manganese complexes amide-bands are the dominating spectral features, just as for the free complexes. Evidence for one or another way of coordination comes from the analysis of the amide modes. The amide I bands, attributed to CO-absorption bands in the range 1650-1630 cm for secondary amides, are visible for most of the Mn(bpR)-Y samples. The amide II band is a combination of C-N stretch and C-N-H bending. 

An example of this approach to protein analysis is illustrated by Figure 6.2c, which shows the amide I band of the enzyme lysozyme in D2O. The amide I band of this protein shows nine component bands. The band at 1610 cm is due to amino acid side-chain vibrations and does not contribute to the amide I band. The relative areas of the amide I components are listed in Table 6.2e, and these may be assigned to the various types of secondary structures. The bands at 1623 and 1632 cm... 

Infrared spectroscopy has been used for the analysis of mixtures of fatty amides (Kauffman, 1964). In dilute chloroform solutions, the amides CH3(CH2) CONH2 absorb consistently at 1681cm and do not display apparent association or enolization. The concentration of unsubstituted amides was quantitatively related to the intensity of an amide I band throughout the range from 1 to 1(X)%. With scale expansion the sensitivity of the method may be extended to 0.03%. 

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