Protein secondary amide bands

Due to limitations in signal-to-noise ratio available for the then common dispersive IR instruments, peptide and protein vibrational spectroscopic studies shifted to emphasize Raman measurements in the 1970s 29-32 Qualitatively the same sorts of empirical correlations as discussed above have been found between frequencies of amide bands in the Raman and secondary structure. However, due to the complementary selection rules for Raman as compared to IR and to the multi-component nature of these polymeric spectral bands, the... 

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 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. 

These kinetics studies required development of reproducible criteria of subtraction of foe H-O-H bending band of water, which completely overlaps foe Amide I (1650 cm 1) and Amide II (1550 cm"1) bands (98). In addition, correction of foe kinetic spectra of adsorbed protein layers for foe presence of "bulk" unadsorbed protein was described (99). Examination of kinetic spectra from an experiment involving a mixture of fibrinogen and albumin showed that a stable protein layer was formed on foe IRE surface, based on foe intensity of the Amide II band. Subsequent replacement of adsorbed albumin by fibrinogen followed, as monitored by foe intensity ratio of bands near 1300 cm"1 (albumin) and 1250 cm"1 (fibrinogen) (93). In addition to foe total amount of protein present at an interface, foe possible perturbation of foe secondary structure of foe protein upon adsorption is of interest. Deconvolution of foe broad Amide I,II, and m bands can provide information about foe relative amounts of a helices and f) sheet contents of aqueous protein solutions. Perturbation of foe secondary structures of several well characterized proteins were correlated with foe changes in foe deconvoluted spectra. Combining information from foe Amide I and m (1250 cm"1) bands is necessary for evaluation of protein secondary structure in solution (100). 

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... 

The amide I mode is most widely used in studies of protein secondary structure [10, 108]. This mode gives rise to infrared band(s) in the region between 1600 cm-1 to 1750 cm 1 and is predominantly due to the CO stretching mode. The major factor responsible for conformational sensitivity of the amide I bond is coupling between... 

The combination of infrared spectroscopy and hydrogen-deuterium exchange is a powerful technique for revealing small differences in protein secondary structure. Few proteins are composed solely of one type of structure, therefore several amide I and amide II frequencies may contribute to any amide I and II band. It is often difficult to resolve all of these frequencies in the difference spectrum, since some of the peaks have bandwidths which are smaller than the amide I or amide II bandwidth and are thus effectively hidden within the main peak. To resolve overlapping bands, second derivative spectra may be generated using a computer programme. The resultant spectrum is presented as absorbance/(wavenumber)2 versus wavenumber. 

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). 

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. 

The overlay of the normahzed spectra indicated that lipid-associated bands near 1070, 1230, 1740, 2852 and 2925 cm had increased. In summary, the relative intensity ratios of Hpid to protein bands decreased in the order (C) > (A) > (B), consistent with the removal of Hpids by toluene and with the removal of proteins by water. Furthermore, the shapes of the amide bands changed, which was consistent with the secondary structure changes in proteins. 

The protein secondary structure of silk fibroin [60] was studied with near-lR spectroscopy, using silk fibers that had been very carefully selected from naturally generated fibers. The isolation of individual fibers allowed the trapping from Nature of a protein with a particular secondary structure. A spider is able to generate different fibers for different uses, with each fiber having its own secondary structural composition. In the case of silk, an individual fiber may well have a particular composition secondary structure, and in this case it is possible to use near-lR spectra to perform a characterization. This is quite remarkable because the use of a relatively prominent amide-1 band in the mid-lR represents a major challenge. 

A rapid FTIR method for the direct determination of the casein/whey ratio in milk has also been developed [26]. This method is unique because it does not require any physical separation of the casein and whey fractions, but rather makes use of the information contained in the whole spectrum to differentiate between these proteins. Proteins exhibit three characteristic absorption bands in the mid-infrared spectrum, designated as the amide I (1695-1600 cm-i), amide II (1560-1520 cm-i) and amide III (1300-1230 cm >) bands, and the positions of these bands are sensitive to protein secondary structure. From a structural viewpoint, caseins and whey proteins differ substantially, as the whey proteins are globular proteins whereas the caseins have little secondary structure. These structural differences make it possible to differentiate these proteins by FTIR spectroscopy. In addition to their different conformations, other differences between caseins and whey proteins, such as their differences in amino acid compositions and the presence of phosphate ester linkages in caseins but not whey proteins, are also reflected in their FTIR spectra. These spectroscopic differences are illustrated in Figure 15, which shows the so-called fingerprint region in the FTIR spectra of sodium caseinate and whey protein concentrate. Thus, FTIR spectroscopy can provide a means for quantitative determination of casein and whey proteins in the presence of each other. 

A considerable amount of work on correlations between the optical activity of the far ultraviolet peptide amide bands (n—n near 222 nm and n—n near 206 and 190 nm) and ordered structures in proteins and polypeptides has accumulated over the past 10—15 years. A significant advance in the evaluation of secondary protein structure has been made by application of the Moffitt equation [Ref. (7)] to the ORD of protein systems in solution ... 

The characteristic bands of the amide groups of protein chains are similar to the absorption bands exhibited by secondary amides in general, and are labelled as amide bands. There are nine such bands, and these are called amide A, amide B and amides I-VII, in order of decreasing frequency these bands are summarised in Table 6.2a. Some of these bands are more useful for conformational studies than others, and the amide A, amide I and amide II bands have been the most frequently used. Knowledge of peptide bond vibrations is basei mainly on normal coordinate calculations using A-raethylacetamide as a model compound. 

The best method to use for the estimation of protein secondary structure involves band-fitting the amide I band. The parameters required, and the number of component bands and their positions are obtained from the resolution-enhanced spectra. The fractional areas of the fitted component bands are directly proportional to the relative amounts of structure that they represent. The percentages of helices, -structures and turns are estimated by addition of the areas of all of the component bands assigned to each of these structures and then expressing the sum as a fraction of the total amide I area. The... 

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 ... 

Secondary amides have a free N—H stretching band in the range 3470-3400 cm" in dilute solutions. The frequency of bonded NH absorptions depends on the nature of the solvent and upon the concentration. With increasing concentration, two bonded NH bands are found at 3340-3140 and 3100-3060cm" (Randall et a/., 1949 Clarke et al, 1949 Richards and Thompson, 1947 Darmon and Sutherland, 1949). The spectra of polypeptides and proteins also have these bands. Two bands that appear as a doublet in secondary amides are due to cis- and trans-rotational isomers containing the free N—H stretching band (Russell and Thompson, 1956). 

GJ. Thomas, Jr., B. Prescott, and D.W. Urry, Raman Amide Bands of lype-II P-Tums in Cyclo-(VPGVG)3 and Poly(VPGVG), and Implications for Protein Secondary Structure Analysis. Biopolymers, 26,921-934,1987. 

The key feature that allows IR spectroscopy to be used to study proteins is the dependence of the amide band on the protein secondary structure (a-helix, parallel and antiparallel j6-sheets, /S-tums, and random). The frequency-structure correlations have been most reliably established for the amide I band (Table 7.9), althongh a nnmber of exceptions to these correlations have already been reported [748, 803], and the assignment for parallel and antiparallel j0-sheets is still debatable [751, 804]. Similar data for amide II bands are less well understood and hence less nseful [803]. Being of lower intensity but free from interference with water (see below), the amide III band is particularly attractive for structural studies [805-812]. A number of comprehensive recent reviews contain more detailed information on the amide band assigmnent [748, 749, 802, 803, 811, 813-817]. 

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