Vibrational Spectroscopy of Polypeptides

Modern vibrational spectroscopy of polypeptides and proteins, as outlined in the previous pages, has made a significant initial contribution as a tool for the detailed analysis of conformation in such molecules. Yet much more remains to be done, both with respect to further refinements in the inputs to the normal-mode calculations as well as in applications to the many general and specific structures that need to be studied. We consider below only briefly some aspects of such future developments. 

The aim of this review is to present these recent developments in the vibrational spectroscopy of peptides, polypeptides, and proteins. We will first discuss the necessary basic aspects of normal-mode calculations. We will then give results for those polypeptide secondary structures that have been studied to date, with an evaluation of the insights obtained from these analyses. Finally, we will comment on the preliminary studies being done on proteins and the prospects for the future. 

Krimm S, Bandekar J. Vibrational spectroscopy and conformation of peptides, polypeptides and proteins. Adv Protein Chem 1986 38 181-365. 

Two-Dimensional Coherent Infrared Spectroscopy of Vibrational Excitons in Polypeptides... 

Because our research is focused on problems relevant to secondary structure of proteins in solution, this section will briefly review the recent developments in spectroscopic techniques applied to this problem. These techniques are considered low-resolution methods which provide global insight into the overall secondary structure of proteins without being able to establish the precise three-dimensional location of individual structural elements [707], Vibrational spectroscopy has played a pioneering role in studying the conformations of peptides, polypeptides, and proteins [702]. The advent of stable and powerful lasers has led to the development of Fourier transform methods which allows the use of powerful computational techniques for the analysis of spectral data [10,103,104], Laser... 

Vibrational Spectroscopy and Conformation of Peptides, Polypeptides, and Proteins... 

In the case of m=2 in dimethylsulfoxide solution, the right-handed a-helical main chain conformation has been confirmed by the circular dichroism in the vibrational region (VCD). (A preliminary result of joint research with Professor T, Keiderling, University of Illinois at Chicago). By the VCD spectroscopy one can obtain information on the main chain conformation of polypeptides without any interference by the side-chain chromophores ( ). The VCD couplet... 

H.D. Middendorf, R.L. Hayward, S.F. Parker, J. Bradshaw A Miller (1995). Biophys. J., 69, 660-673. Vibrational neutron spectroscopy of collagen and model polypeptides. 

Previous infrared studies indicate that proteins with a large amount of 3-sheet structure absorb near 1240 cm and those with a-helix stucture absorb near 1280 cm l (22,32). Absorbances for denatured albumin, reportedly containing random and 3-sheet conformations, are found at 1240 and 1260 cvT (22). These assignments correlate with the more studied Raman spectroscopy of the amide III region which has vibrations at 1230-1250 cm l for 3-sheet structure, at 1260-1290 cm for a-helix structure, and at 1240-1265 cm l for unstructured polypeptide (34,35). 

Raman spectroscopy is a vibrational spectroscopic technique which can be a useful probe of protein structure, since both intensity and frequency of vibrational motions of the amino acid side chains or polypeptide backbone are sensitive to chemical changes and the microenvironment around the functional groups. Thus, it can monitor changes related to tertiary structure as well as secondary structure of proteins. An important advantage of this technique is its versatility in application to samples which may be in solution or solid, clear or turbid, in aqueous or organic solvent. Since the concentration of proteins typically found in food systems is high, the classical dispersive method based on visible laser Raman spectroscopy, as well as the newer technique known as Fourier-transform Raman spectroscopy which utilizes near-infrared excitation, are more suitable to study food proteins (Li-Chan et aL, 1994). In contrast the technique based on ultraviolet excitation, known as resonance Raman spectroscopy, is more commonly used to study dilute protein solutions. 

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). 

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). 

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