Infrared spectroscopy protein secondary structure determination

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... 

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. 

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. 

Very few of the infrared studies of proteins have been carried out on aqueous solutions of the proteins. Except for the work of Koenig and Tabb (1), the few aqueous IR studies have been on single proteins. Correspondingly, most of the assignments of the backbone vibrations (the so-called Amide I, II, III, etc. vibrations) have been based on either Raman spectra of aqueous solutions (2) or on infrared spectra of proteins in the solid state (3). Where infrared solution spectra have been obtained, it has mostly been on D2O solutions (4) - not H2O solutions. Since these Amide I, II, III, etc. vibrations involve motion of the protein backbone, they are sensitive to the secondary structure of the protein and thus valid assignments are necessary in order to use infrared spectroscopy for determining the conformations of proteins. 

Fourier-transformed infrared (FTIR) is another excellent method to study protein folding. Unlike the well-known use of FTIR as a method for the identification of functional groups, in terms of protein structure this method allows the determination of secondary structure. The frequency of vibration of the amide I band of the peptide chain (1500-1600 cm M heavily depends on the structure of the protein. FTIR has the advantage of being more sensitive for the study of proteins that contain (3-sheet elements as compared to CD. Furthermore, since FTIR spectroscopy can be applied to solids also, it allows the structural analysis of aggregated protein deposits. The availability of the rapid step-scan method for FTIR is also very useful for the study of rapid folding reactions (see Vibrational Spectroscopy). 

Kumosinski, T.F. Farrell, H.M., Jr. Determination of the global secondary structure of proteins by Fourier transform infrared (FTIR) spectroscopy. Trends Food Sci. Technol. 1993, 4, 169-175. 

Because neither PrP" nor PrP " " could be crystallized, it was not possible to establish the 3D structure of these proteins by X-ray crystallography To circumvent this xmfavorable situation, Prusiner used circular dichroism and Fourier transformed infrared (FTIR) spectroscopy, both techniques that allow determination of the respective percentages of the secondary structures (a, (3, and turns) in a peptide or a protein. Applied to PrP and PrP samples, these spectroscopic approaches definitely demonstrated that both isoforms have a distinct 3D structure. In a seminal article published in 1993, the Prusiner s group established that PrP is chiefly an a-helical protein (43% of a-helix structure) with few -structures (3%). In contrast, PrP has less a-helix (30%) but has gained a high percentage of -structures (43%). 

This process was confirmed by both circular dichroism and Fourier transform infrared spectroscopy, suggesting tirat a-synuclein has a slightly collapsed conformation, with 14% of its secondary structure corresponding to tums. In line with these data, the determination of the hydrodynamic dimensions of a-synuclein revealed that the protein was indeed... 

We have recently used polarized infrared (IR) spectroscopy to examine the extent and organization of protein structures in membrane associated proteins (4-6). In particular, with membrane reconstituted RC, we "ave determined the net orientation of thea -helical segments with respect to the membrane plane (6). In this article, we have made a comparison of the protein structures in the LMH and LM complexes in order to analyze the composition and the orientation of the secondary structures localized within the LM complex. We have investigated the UV circular di-chroism (CD) and polarized IR spectra of the LMH and LM complexes reconstituted in lipid vesicles. Our results show that transmembrane a-helices are present in both LMH and LM complexes. In addition, we also discussed some general features of membrane IR dichroism spectra. 

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