Polypeptides structure, infrared analysis

Infrared Analysis of the Structure of Amino Acids, Polypeptides, and Proteins... 

Infrared analysis of the structure of amino acids, polypeptides and proteins. Advanc. Protein. Chem. 7, 291—318 (1952). 

It seems to the reviewer that more fundamental experimental and theoretical work must be done on the interpretation of the spectra of the NH frequencies in amides and polypeptides, especially in relation to hydrogen bonding and crystalhne structure, before categorical statements can be made regarding protein structure by infrared analysis. 

Solid films have been examined frequently for infrared analysis in biochemical work, for example, in structural studies of proteins, polypeptides, and polysaccharides. Such films have been of particular value for studying polarization spectra of macromolecules in intact films and in oriented ones (stretched, rolled, or stroked), thereby permitting knowledge to be gained concerning spatial arrangements within the molecule and conformational effects among molecules. (See The Use of Polarized Infrared Radiation and the Measurement of Dichroism, p. 73, for a detailed discussion.) A few workers have discussed the film technique (Lecomte, 1948 Randall et al., 1949 Hacskaylo, 1954). 

Table 5.8 Polypeptides detected during the LC-electrospray-MS analysis of the tryptic digest from /Mactoglobulin ifiLG). Reprinted from J. Chromatogr., A, 763, Turula, V. E., Bishop, R. T., Ricker, R. D. and de Haseth, J. A., Complete structure elucidation of a globular protein by particle beam liquid chromatography-Fourier transform infrared spectrometry and electrospray liquid chromatography-mass spectrometry - Sequence and conformation of /Mactoglobulin , 91-103, Copyright (1997), with permission from Elsevier Science...

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). 

Quantification of ORD and CD Data. In principle ORD and CD can be used to calculate the amounts of a, / , and random conformations in protein, but in practice such estimates are subject to large errors. The Moffitt-Yang plot is probably the best estimate of percentage of a-helicity, but it is unable to distinguish between the ft and random structures. A detailed analysis of CD bands and their resultant Cotton effects, combined with infrared data, is the most promising approach even here the limits of error are large (82). Traditional estimates have been based on combinations of a-helix and random coil, and attention has been centered upon estimation of helical content. Consideration of j3 structure has been introduced more recently. The technique must be calibrated empirically with synthetic polypeptides of known conformation, and the proper choice of reference is not obvious. The /3 structure seems to be particularly variable in its rotational properties (27, 82). 

Pioneering work by the Alix laboratory on the secondary structure of human elastin and the solubilized K-elastin, estimated the molecule to be composed of 10% a-helices, 35% P-strands and 55% undefined conformation. These estimations were based on Fourier transform infrared (FTIR), near infrared Fourier transform Raman spectroscopy and circular dichroism (CD) (15). To further investigate the nature of the elasticity, polypeptides of hydrophobic sequences containing exons 3, 7, and 30 of human elastin were analyzed by CD and Classic Raman spectroscopy, revealing polyproline II (PPII) helix secondary structures in both the aqueous and solid phase. Further analysis of exon 30 by FTIR spectroscopy determined that this sequence was characterized by both PPII as well as p-sheets structures (15). The presence of these structures were dependent on temperature, concentration and / or time, where lower temperatures and concentrations favored the PPII structure and higher temperatures and concentrations favored p-sheets (16). 

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