Infrared spectroscopy protein secondary structure

The conformational changes which have been described so far are probably all relatively small local changes in the structure of H,K-ATPase. This has been confirmed by Mitchell et al. [101] who demonstrated by Fourier transform infrared spectroscopy that a gross change in the protein secondary structure does not occur upon a conformational change from Ei to 3. Circular dichroism measurements, however [102,103], indicated an increase in a-helical structure upon addition of ATP to H,K-ATPase in the presence of Mg and... 

Surewicz, W.K., Mantsch, H.H., Chapman, D. (1993). Determination of protein secondary structure by Fourier transform infrared spectroscopy A critical assessment. Biochemistry, 32, 389-394. 

Since infrared spectroscopy also provides information about physical structure, infrared imaging can be used to determine spatial distribution of physical properties as well. Some of the properties include intermolecular and intramolecular order, hydrogen bonding, protein secondary structure, complexation and functional group orientation. 

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... 

Prestrelski SJ, Byler DM, Liebman MN. Generation of a substructure library for the description and classification of protein secondary structure. II. Application to spectrastructure correlations in Fourier transform infrared spectroscopy. Proteins 1992 14 440-450. 

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. 

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. 

Infrared spectroscopy is largely unaJTected by the aforementioned problems. The technique can be used to study the secondary structure of proteins, both in their native environments, as well as after reconstitution into model membranes. In particular, infrared spectroscopy offers several advantages in studies of protein-lipid interactions. Information about lipid conformation and protein secondary structure can be obtained in a single experiment from the same sample. 

Table 7.4 Characteristic amide I band assignments of protein secondary structures. From Stuart, B., Biological Applications of Infrared Spectroscopy, ACOL Sales,...

Fourier transform infrared spectroscopy (FTIR) is a powerful tool used to monitor changes in protein and polypeptide secondary structure during processing. After exposure of a protein to infrared light, its secondary structure can be determined from the spectra obtained from the absorption of different wavelengths corresponding to specific vibration frequencies of the amide bonds (Jackson and Mantsch, 1995a). 

To summarize, from ouf data, it can be concluded that in both LMH and LM complexes, all the protein secondary structures are oriented almost to the same extent. This observation suggests a model of the RC in which all three subunits L, M, H, have transmembrane a-helices. The observed differences between LMH and LM in the absence of nujol could be due to differences i) in the state of hydration in LMH and LM, ii) in the optical properties of the multilayers, iii) in oriented secondary structures (ex g-structure) which are removed with nujol. Similar findings were observed on other oriented air-dried membranes such as chro-matophore, thylakoid and purple membrane. We find that distortions of the amide I and amide II absorptions brought about by dehydration and/ or optical reflection effects are a general feature of protein infrared spectra. These effects are more evident in samples with small extents of dichroism (eg. air-dried samples of soluble proteins such as cytochrome c or ribonuclease) since these effects are present to the same extent, but largely hidden in samples with large dichroic signals (eg. oriented purple membrane). These results indicate that a more precise determination of membrane protein orientation by polarized IR spectroscopy requires these effects to be taken into account. 

The availability of the purified transporter in large quantity has enabled investigation of its secondary structure by biophysical techniques. Comparison of the circular dichroism (CD) spectrum of the transporter in lipid vesicles with the CD spectra of water-soluble proteins of known structure indicated the presence of approximately 82% a-helix, 10% ) -turns and 8% other random coil structure [97]. No / -sheet structure was detected either in this study or in a study of the protein by the same group using polarized Fourier transform infrared (FTIR) spectroscopy [98]. In our laboratory FTIR spectroscopy of the transporter has similarly revealed that... 

Vandenbussche G, Clercx A, Clercx M, et al. Secondary structure and orientation of the surfactant protein SP-B in a lipid environment. A Fourier transform infrared spectroscopy study. Biochemistry 1992 31(38) 9169-9176. 

Fourier transform infrared/photoacoustic spectroscopy (FT-IR/PAS) can be used to evaluate the secondary structure of proteins, as demonstrated by experiments on concanavalin A, hemoglobin, lysozyme, and trypsin, four proteins having different distributions of secondary... 

Most of the above membrane-oriented studies were carried out for peptides in multilayer systems that were collapsed or transferred onto a sample cell surface. An alternative and very interesting way to study membrane systems is by IRRAS (infrared reflection absorption spectroscopy) at the air-water interface. In this way, unilamellar systems can be studied as a function of surface pressure and under the influence of various membrane proteins and peptides added. Mendelsohn et al.[136] have studied a model series of peptides, [K2(LA) ] (n = 6, 8, 10, 12), in nonaqueous (solution), multilamellar (lipid), and unilamellar (peptide-IRRAS) conditions. In the multilamellar vesicles these peptides are predominantly helical in conformation, but as peptide only monolayers on a D20 subphase the conformation is (1-sheet like, at least initially. For different lengths, the peptides show variable surface pressure sensitivity to development of some helical component. These authors further use their IR data to hypothesize the existence of the less-usual parallel (i-sheet conformation in these peptides. A critical comparison is available for different secondary structures as detected using the IRRAS data for peptides on H20 and D20 subphasesJ137 ... 

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). 

Following successful recovery of peptide/protein molecule from the microspheres, a simple spectrophotometric method does not always allow discrimination between the monomeric protein form and its aggregates. However, HPLC might separate these species and thus provides more accurate qualitative data [96], But HPLC cannot quantify exclusively the amount of active protein antigen, as is the case with ELISA techniques [97], Nowadays, Fourier transform infrared (FTIR) spectroscopy has become a popular, noninvasive method, as it is able to characterize the secondary structure of entrapped proteins [26, 95, 98-101], Only recently, the integrity of their primary structure was evaluated, thanks to a new matrix-assisted laser... 

The secondary structure of proteins may also be assessed using vibrational spectroscopy, fourier transform infrared spectroscopy (FTIR), and Raman spectroscopy both provide information on the secondary structure of proteins. The bulk of the literature using vibrational spectroscopy to study protein structure has involved the use of FTIR. Water produces vibrational bands that interfere with the bands associated with proteins. For this reason, most of the FTIR literature focuses on the use of this technique to assess structure in the solid state or in the presence of non-aqueous environments. Recently, differential FTIR has been used in which a water background is subtracted from the FTIR spectrum. This workaround is limited to solutions containing relatively high protein concentrations. 

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