The amide III band

In 3, the amino functional group is two methylene units removed from the ferrocene nucleus. It appears from the instantaneous and quantitative formation of h from 3 that this feature minimizes steric effects and also enables 3 to undergo the Schotten-Baumann reaction readily without the classical a-metallocenylcarbenium ion effects providing any constraints. The IR spectrum of showed the characteristic N-H stretch at 3320 cm" (s), the amide 1 (carbonyl) stretch at 1625 an - -(s), the amide II (N—H) stretch at 1540 cm (s), and the amide III band at 1310 cm 1(m). In addition, characteristic absorptions of the ferrocenyl group were evident at 1100 and 1000 cm l (indicating an unsubstituted cyclopentadienyl ring) and at 800 cm"l. 

Both amide I and amide III bands are seen in Raman spectra of proteins.30 Lippert et al. devised the following method for estimating the fractions of a-helix, (3 sheet, and random coil conformations in proteins.31 The amide I Raman bands are recorded at 1632 and 1660 cm 1 in DzO (amide I ). The amide III band, which is weak in DzO, is measured at 1240 cm-1 in H20. The intensities of the three bands relative to the intensity of an internal standard (the 1448 cm 1 CH2... 

For verifying how the temperature affects the structure of the adsorbed molecules so that the direction of their dipole moment flips, we measured the infrared spectrum of an LC film at two temperatures as shown in Fig. 7. The peaks in Fig. 7a are of the amide-I (1,668 cm ) and amide-II (1,544 cm ) bands and those in Fig. 7b are of the amide-III (3,200-3,500 cm ) band (N-H stretching). Clearly a dramatic change occurs in the vibrations of the polypeptide upon cooling of the sample. The amide-I vibration is split and shifted to lower wavenumber. The amide-III band also splits but in addition it is shifted to higher wavenumbers. 

Yu (1974, 1977) also showed, by comparison of the environments of the peptide backbone, as reflected in the amide III bands, that there was virtually no difference between the spectra of crystals of lysozyme and a-lactalbumin and their respective solutions. However, the spectra were altered by lyophilization, with respect to main-chain conformations (this is considered again in Section XI). 

The amount of N—H deformation in each band is suggested by the factor vn/ro, where vo refers to the corresponding band in the A -deuterated compound. For the gas state these shifts are 1.002, 1.074, and 1.356 (1422). The obvious interpretation is that the large deuteration shift of the amide III band identifies it as the deformation mode, Vft. This conclusion is warranted, however, only after normal coordinate analysis confirms the significance of vh/j d- Miyazawa, Shimanouchi, and Mizushima have made such calculations, and a portion of their results are presented in Table 3-XVII. The boldface numbers in this table show that Ub contributes substantially to both the amide II and III bands and that these vibrational modes are best described as characteristic of the C—N—H group. (The large deuteration shift of the amide III band must be attributed to mechanical interactions.)... 

Fig. 10. IR spectra of the wild-type E. coli LamB synthetic signal peptide in phospholipid monolayers. (A) Peptide adsorbed to the monolayer (film formed above the critical insertion pressure of the peptide). (B) Peptide adsorbed and inserted (film formed below the critical insertion pressure). Characteristic amide I bands for a-helix (or random coil) 1660 era", for -structure 1630 era. The amide III band (at lower frequencies) was used to confirm that the 1660 cm band was due to helix and not coil. Experimental details are reported in Briggs et al. (1986). Copyright 1986 by the American Association for the Advancement of Science.

Homogeneous 1 2 and 1 4 molar mixtures of aliphatic Urethanes III or IV with polypropylene glycol (2000 MW) are similar to the aromatic Urethane I or II mixtures in the Amide I and II regions. However, the Amide III band absorbs at 1245 cm-1, with a weaker 1260-cm 1 shoulder. The 1210-cm 1 shoulder due to vibrations of the aromatic ring is missing. Also, the 1320-cm 1 shoulder present in the aromatic mixtures is absent in the aliphatic mixtures. 

B. The frequency of the Amide III bands always indicates the presence of 3-sheet and only indicates the presence of... 

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]. 

Liu et al. [43] were the first to present spectra, using a NIR-FT-Raman spectrometer of normal and cancerous tissues of the gynecologic tract. Spectral differences between normal tissue and benign and malignant tumor tissue of the cervix, uterus, endometrium, and ovary were found in the CH-deformation band at 1445 cm the Amide III band at 1262 cm and the Amide I band at 1659 cm ... 

The infrared spectra of proteins and polypeptides comprise essentially four strong vibrational modes associated with the peptide link. These are the Amide A band near 3300 cm (vn-h)> the Amide I band near 1650 cm (vc=o), the Amide II band near 1550 cm " and the Amide III band near 1250 cm The latter two modes are both associated with combined stretch-... 

The vibrational spectra of reference material are introduced in Figure 3.1, which belong to the main components of soft tissue. The IR spectrum (trace A) and Raman spectrum (F) of the all-beta protein concanavalin A are shown in Figure 3.1. IR bands due to the peptide backbone with P-sheet secondary structures are found at 3284 (amide A), 1636 (amide I), 1531 (amide II), and 1235 cm-i (amide III). Bands at 1403 (COO ) and 2963, 2874, 1455 cm- (CHg) are assigned to amino acid side chains. These bands are located in the Raman spectrum at similar positions at 1398 and 1449 cm . The Raman amide I band is centered at 1672 cm , the amide III band at 1238 cm , and the weak amide II band is not observed. Instead, other Raman bands of amino acids are identified at 759 and 1555 cm for Trp 621,1003, 1031, and 1208 cm for Phe 643, 829, and 853 cm for Tyr and 1126, 1317, and 1340 cm (CH2/CH3) for aliphatic amino acids. The IR spectrum (B) and Raman spectrum (G) of the all alpha protein bovine serum albumin show a number of differences. The amide bands... 

Effects of sample orientation in Raman microspectroscopy of collagen fibers and their impact on the interpretation of the amide III band. Vih. Spectrosc.,... 

Unfortunately, for some proteins, the amide I for the a-helix may be hidden by strong absorptions by water, in which case the amide III band may be investigated. Changes may also be observed in the C-C stretching vibrations that occur in the region 1000-945 cm (10.00-10.58pm). 

The spectra of proteins found in cells have a strong amide I band near 1650cm" ( 6.06 im). This band is affected by the environment of the peptide linkage and the protein s secondary amine. The amide II band occurs near 1530 cm" ( 6.54pm) and the amide III band occurs near 1245 cm" ... 

Raman spectroscopy is not yet commonly used to follow conformational variations of proteins. Only a few reports are available. Brunner and Sussner (1972) and Brunner and co-workers (1974) studied the thermal denaturation of bovine pancreatic trypsin inhibitor (BPTI) with Cys 14-Cys 38 bond reduced and with this reduced molecule then carboxamidomethylated. The native molecule was first analyzed. The amide I band at 1664 cm which is characteristic of p structure indicates that this structure is dominant in the molecule. The amide III band displays three well resolved peaks at 1243, 1265, and 1288 cmwhich are characteristic of antiparallel P sheets, random coil, and a helical structures. From these data Brunner and Sussner (1972) and Brunner and co-workers (1974) evaluated the content of secondary structures to be ca. 45% P structure, 30% random coil, and 25% a helix this is in satisfactory agreement with the X-ray data. 

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