Paul's Practical Guide - page 6
Structure Analysis of Proteins
CD spectra in the far-UV region (170-260 nm) provide important information about protein secondary structure. Common secondary structure motifs exhibit predictable CD spectra (See Figure 1). Based on these and the spectra of standard proteins, there are many algorithms currently available for protein secondary structure analysis. The one that is available in GlobalWorks is based on the algorithm shown in Analytical Biochemistry1. This algorithm uses a basic set of sixteen model protein structures. The shape of the spectrum is compared to the basis spectra and five structural contributions are extracted (alpha-helix, parallel beta-sheet, antiparallel beta-sheet, beta turns, and other). To collect data for this analysis, a rather rigid format must be adhered to. The data must start at 260 nm and span 2 nm per data point, and the data must end at 184, 182, 180, or 178 nm (38-41 data points). No information about protein concentration is required. There are several websites in which other fitting algorithms can be obtained2. There are also several reviews of secondary structure prediction algorithms3.
If the data are recorded in molar ellipticity, the alpha-helical content can be estimated from the molar ellipticity at 222 nm by Equation 14:
% alpha-helix = (-[ ]222 nm + 3000)/39000
Changes in protein tertiary structure can be observed using CD. The wavelength range most sensitive to tertiary structure changes is the near UV region of the spectrum. CD signals in this region originate from aromatic side chains, and unlike the relatively predictable far-UV, signals in this region are more sensitive to tertiary conformational changes, which alter the environment of the aromatic side chains. This is in contrast to the far-UV in which changes in secondary structure are required to change the CD signal originating from the peptide background. For proteins containing chromophores, such as heme proteins, the chromophore can act as a tertiary structure probe. For example, the heme in hemoglobin only exhibits a CD signal only when in an asymmetric environment (i.e. bound to a folded protein). Thus the CD signal will act as a probe of protein tertiary structure.
Thermal and Chemical Denaturations of Proteins
One of the most useful applications of CD in the study of proteins is monitoring protein denaturations, which can be initiated either thermally or chemically. In the experiment, CD data are collected as a function of temperature or denaturant concentration. Data can be collected at a single wavelength, resulting in two-dimensional denaturation curves, in which CD signal is recorded versus temperature or denaturant concentration, These curves are fit by known denaturation models to give information about protein structure and stability. Additionally, CD spectra can be collected as a function of temperature or denaturant, resulting in a three-dimensional data set with the axes being wavelength, temperature or concentration, and CD intensity. The advantage of this is that data from many wavelengths are included in the subsequent fitting procedures. This 3D data set should be passed though singular value decomposition (SVD) to determine the number of species involved in denaturation and to remove the noise so that the data can be better fitted to unfolding mechanisms to obtain thermodynamic information.
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Footnotes
1) Compton, L.A. and Johnson, C.W., Analytical Biochemistry. 155-167, 1986.
2) http://www2.umdnj.edu/cdrwjweb, http://www.cryst.bbk.ac.uk/cdweb/html/home.html, http://akilonia.cib.csic.es/~pablo/K2D/
3) Greenfield N.J., Analytical Biochemistry. 235, 1-10, 1996.
Sreerama, N., and Woody, R.W., Analytical Biochemistry. 282, 243-251, 2000.
4) Morrow, J.A., Segall, M.L., Lund-Katz, S., Phillips, M.C., Knapp, M., Rupp, B. and Weigraber, K.H. Biochemistry. 39, 11657-11666, 2000.
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