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4.2 Identification without 3D Structure
4.2.3 FT-infrared spectroscopy
Like circular dichroism analyses of proteins, Fourier transform infrared
(FT-IR) spectroscopic studies and are easily performed and require relatively
small amounts of material (~0.1 mg). The infrared spectra of polypeptides
exhibit a number of so-called amide bands which represent different vibrational
modes of the peptide bond. Of these, the amide I band is most widely
used for secondary structure analyses. The amide I band results from the
C=O stretching vibration of the amide group coupled to the bending of the
N-H bond and the stretching of the C-N bond. These vibrational modes, present
as infrared bands between approximately 1600-1700 cm-1, are sensitive to
hydrogen bonding and coupling between transition dipole of adjacent peptide
bonds and hence are sensitive to secondary structure.
A critical step in the interpretation of IR spectra of proteins
is the assignment of the amide I component bands of different types of
secondary structure. Amide I bands centered around 1650-1658 cm-1 are generally
considered to be characteristic of alpha helices. Unordered structure and
turns also give rise to amide I bands in this region complicating analyses.
Beta sheets give rise to highly diagnostic bands in the region 1620-1640
cm-1. Parallel and antiparallel beta strands are distinguishable only as
antiparallel strands contain a large splitting of the amide I band due
to the interstrand interactions. Water (H2O) also has an intense IR band
in the region of the amide I band and requires that samples are measured
in 2H2O or that the solvent resonance is subtracted (digitally). Subtraction
of the H2O band can lead to artifacts hampering interpretation.
Secondary structure from FT-IR spectra
Numerous attempts have been made to extract quantitative information on
protein secondary structure from analyses of these amide I bands (for reviews
see Byler & Susi, 1986;Surewicz, et al, 1993). Both curve-fitting and
pattern recognition techniques have been applied with varying success.
Since the potential sources of error in CD and FT-IR analyses of secondary
structure content are largely independent, the two methods are highly complementary
and could be used in conjunction to increase accuracies.
Despite limitations in the quantitative assessment of protein
secondary structure content, FT-IR (like CD) provides a good tool too monitor
conformational changes in polypeptides and proteins.
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Secondary structure from FT-IR spectra
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No Title - 31 MAY 96
written by Kurt D. Berndt
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