Proteins amide I band

The plots of the intensities of selected characteristic bands as a function of lateral position (so-called chemical maps) provide information on the amount of the respective molecules or molecular groups in the different morphological structures (Fig. 4.2). The band at 784 cm 1 can be assigned to out-of-plane deformation vibrational modes of the nucleobases cytosine, thymine and uracil and serves as an indicator for the presence of nucleic acids. At 483 cm-1, a C-C-C deformation of carbohydrate polymers such as starch or pectin is present in some of the spectra. To study the distribution of protein compounds, we analysed characteristic signals of the amino acid phenylalanine (1002 cm 1 ring breathe) as well as of the protein amide I band (1651 cm-1) that is brought about by vibrations of the protein backbones. The maximum of the phenylalanine signal co-localizes with a maximum in protein content... 

This is the fundamental deformation band. It overlaps the protein amide I band. This band can be used to monitor changes in water content in a variety of materials including meats, protein isolates, and starch. 

Figure 10.2 Bidimensional correlation maps corresponding to changes in intensity of a band composed of one (bottom) or two peaks (top). Synchronous maps are located at the left and asynchromous to the right. The x- and y-axes correspond to the numbers of the point on the artificial curve and the z-axis is the correlational intensity. Roughly they represent a protein amide I band in a DjO buffer. Negative peaks are shaded...

As observable from figures 5 and 6, the amide specific band absorptions for proteins, amide I band around 1654 cm-i and amide II band around 1541 cm" (Firth et al. 2008 Banuelos et al. 1995) are not changed when LDL was deposited on the gold support. This observation is important because it proved that the secondary structure of protein is preserved subsequent deposition therefore it can be concluded that the deposition on solid support did not affect theLDL functionality, and, consequently, that deposed LDL is expected to react with free radicals according to the same pathway as free LDL. Moreover, it should be mentioned that this argument is consistent with the data published by Paker (Paker, 1991) where it is mentioned that LDL ex vivo peroxidation pathway is similar as in vivo peroxidation pathway. 

To obtain this detailed structural information, it is necessary to enhance the resolution of the protein amide I band, which usually appears as a single broad absorbance contour (Figure 1). The widths of the overlapping component bands are often greater than the separation between the absorbance maxima of neighboring bands. Because the band overlapping is beyond instrumental resolution, several mathematical band-narrowing methods (i.e., resolution enhancement methods) have been developed to overcome this problem [11,50-52,54]. For studies of lyophilization-induced structural transitions, calculation of the second derivative spectrum is recommended [11]. This method is completely... 

V c=o of amides of proteins. Amide I band. May also contain contributions from C=C stretches of olefmic and... 

Figure 6.13 Raman maps of the selected inner endosperm region for wild type (A) and ae mutant maize (B). The maps are overlaid on the visible images. Scale bars = 10 im. Area of protein amide I band (1655 cm" ) in wild type (a) and ae maize (b) map of the 9527942 cm" band ratio for...

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... 

Significantly, the bio-inorganic and polymer-containing PM nanocomposites showed no significant shift in the protein amide I and II vibration bands, or in the characteristic 567 nm optical absorption band of the retinal chromophore of BR, indicating that the structural and dynamical properties of the membrane-bound... 

Both methods are also limited in accuracy of secondary structure determinations because spectral peaks must be deconvolved estimates are made of the overlapping contributions of different structural regions. These estimates may introduce error based on the reference spectra used and because deconvolution methods equate crystallographic secondary structure with the secondary structure of the protein in solution (Pelton and McLean, 2000). As amyloid fibrils are neither crystalline nor soluble, there may be even greater error in estimates of secondary structure. To compound the problem, estimates of /f-sheet content are less reliable than those of a-helix, because of the flexibility and variable twist of / -structure (Pelton and McLean, 2000). In addition, / -sheet and turn bands overlap in FTIR spectroscopy (Jackson and Mantsch, 1995 Pelton and McLean, 2000). Side chains also contribute to spectral peaks in both methods, and they can skew estimates of secondary structure if not properly accounted for. In FTIR spectra, up to 10-15% of the amide I band may arise from side chain contributions (Jackson and Mantsch, 1995). 

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). 

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. 

The spectra of dry films of intact ghosts prepared by lysis in 20 millios-molal phosphate buffer (21) and of ghost protein prepared by cold butyl alcohol extraction (51) are shown in Figure 6. In both cases the amide I band occurs at 1651 cm. 1 and shows no shoulder near 1630 cm. 1, characteristic of fl structure. The amide II band is also unaffected by removal of lipid and occurs at 1540 cm."1. As expected, extraction of lipid results in removal of the band at 1737 cm. 1 assigned to lipid ester carbonyl stretch and a decrease of the band at about 1455 cm."1 arising from methylene and methyl bending. 

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...

If peptide chains can be oriented in a regular fashion, it may be useful to measure infrared linear dichroism.24 25 Absorption spectra are recorded by passing plane polarized light through the protein in two mutually perpendicular directions, with the electric vector either parallel to the peptide chains or perpendicular to the chains. Such a pair of spectra is shown in Fig. 23-3A for oriented fibrils of insulin. In this instance, the insulin molecules are thought to assume a P conformation and to be stacked in such a way that they extend transverse to the fibril axis (a cross-P structure). When the electric vector is parallel to the fibril axis, it is perpendicular to the peptide chains. Since the amide I band is dominated by a carbonyl stretching motion that is perpendicular to the... 

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. 

Protein Amide I/amide II band (cm-1) CD peaks (nm) Specific activity Km y v max... 

While limited changes in the features of the amide I band are observed after BSA adsorption on the two silicas the amide II band, present in the spectrum of the native BSA in solution (curve a) is absent in the spectrum of the protein adsorbed on A50 (curve b), whilst it is partially maintained in that of BSA on Qzm (curve c). This behaviour indicates that the interaction with the surface of the amorphous silicas resulted in an opening of the hydrophobic pockets, allowing the N-H groups therein contained to be converted by contact with D20 in N-D, producing an IR absorption at lower frequency (ca. 1450 cm, not shown). On Qzm the conformational changes are less pronounced. 

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... 

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