Proteins amide vibrations

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... 

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... 

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... 

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). 

Figure 7.10 N N ratio of protein amide II band in two Cladophora glomerata cells showing the subcellular distribution of NOs incorporation, (ca. 150pm diameter). Reproduced with permission from Ref [26] Vibrational Spectroscopy.

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. 

As a first simple example, we apply the above explained methodology to study the vibrational energy redistribution of A-methylacetamide (NMA) in D2O following the laser excitation of the amide I mode in its first excited state. NMA has been used in numerous studies as a model system the peptide bond that links the various amino acids in a protein. The vibrational life time of the C=0 vibration of NMA has been studied experimentally in Ref [58], revealing a typical lifetime on the order of 1 ps (the decay is observed to occur in a biexponential, or nonexponential, manner). The relaxation rate does not change much whether the peptide bond is isolated (i.e., in NMA) or whether it is part of a larger peptide or protein. Furthermore, the decay is hardly affected by temperature, and increases by less then a factor of two when decreasing the temperature below 100 K. 

C=0 - stretching vibration for carbonyl group for protein, amide I 1656 cm-i band correspwnd to amide I helix structure being the result of 60% vibration of C=0 group and 20% vibration of N-H... 

N-H - deformation (scissoring)vibration for amine group of protein, amide II Is due to 60 % vibration of N-H bound and the rest from -CO-NH-functional group... 

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. 

MM2 was, according the web site of the authors, released as MM2 87). The various MM2 flavors are superseded by MM3, with significant improvements in the functional form [10]. It was also extended to handle amides, polypeptides, and proteins [11]. The last release of this series was MM3(%). Further improvements followed by starting the MM4 series, which focuses on hydrocarbons [12], on the description of hyperconjugative effects on carbon-carbon bond lengths [13], and on conjugated hydrocarbons [14] with special emphasis on vibrational frequencies [15]. For applications of MM2 and MM3 in inorganic systems, readers are referred to the literature [16-19]. 

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... 

The wavelengths of IR absorption bands are characteristic of specific types of chemical bonds. In the past infrared had little application in protein analysis due to instrumentation and interpretation limitations. The development of Fourier transform infrared spectroscopy (FUR) makes it possible to characterize proteins using IR techniques (Surewicz et al. 1993). Several IR absorption regions are important for protein analysis. The amide I groups in proteins have a vibration absorption frequency of 1630-1670 cm. Secondary structures of proteins such as alpha(a)-helix and beta(P)-sheet have amide absorptions of 1645-1660 cm-1 and 1665-1680 cm, respectively. Random coil has absorptions in the range of 1660-1670 cm These characterization criteria come from studies of model polypeptides with known secondary structures. Thus, FTIR is useful in conformational analysis of peptides and proteins (Arrondo et al. 1993). 

---