Circular dichroism, protein structure analysis using

After device construction, structural and functional analysis are critical. One might argue that only the second issue matters, but structural data often give insights into why devices perform suboptimally, and provide important clues about how to improve device function. We routinely use protein analytics (matrix-assisted laser desorption-ionization mass spectroscopy, amino acid composition analysis, gel electrophoresis, Western blotting, circular dichroism, vari-... 

Aside from the direct techniques of X-ray or electron diffraction, the major possible routes to knowledge of three-dimensional protein structure are prediction from the amino acid sequence and analysis of spectroscopic measurements such as circular dichroism, laser Raman spectroscopy, and nuclear magnetic resonance. With the large data base now available of known three-dimensional protein structures, all of these approaches are making considerable progress, and it seems possible that within a few years some combination of noncrystallo-graphic techniques may be capable of correctly determining new protein structures. Because the problem is inherently quite difficult, it will undoubtedly be essential to make the best possible use of all hints available from the known structures. 

The possibilities of application of far-UV circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy in analysis of thermal stability of proteins and structural changes within protein molecules as well in explanation of cross reactivity between food allergens have been described in more detail in Section 3.4. Likewise nuclear magnetic resonance (NMR), especially 2D and multidimensional NMR as well as the method based on diffraction of monochromatic x-rays widely used in examination of tertiary structures of allergens have been described in Section 3.4 and by Neudecker et al. (2001) and Schirmer et al. (2005). 

Baumruk V. Pancoska P. Keiderling TA. Prediction of secondary structure using statistical analysis of electronic and vibrational circular dichroism and Fourier transform infrared spectra of proteins in H2O. J Mol Biol 1996 259 774-791. 

One important experimental result was available, the quantitative measurement of the fraction of each secondary structural element by circular dichroism (CD) on purified lipid-protein complexes. This provided a constraint that allowed a careful evaluation of the secondary structure predictions derived from the various approaches, some of which were developed for water-soluble proteins and therefore of uncertain reliability for proteins in a lipid environment. The data from these analyses were combined using an integrated prediction method to arrive at a consensus secondary structure model for each protein. The integrated method involved 36 steps, with independent predictions at each step. The final model was based on an evaluation of the various predictions, with judicious intervention by the authors. As an aid to developing the appropriate weighting of all the data, they carried out the analysis for apoE-3 without reference to the available crystal structure (Wilson et al., 1991), then used the known structure of the HDL-binding amino-terminal domain of apoE-3 as feedback to reevaluate the weighting. 

FT-IR spectroscopy is particularly useful for probing the structure of membrane proteins. Until recently, a lack of adequate experimental techniques has been the reason for the poor understanchng of the secondary structure of most membrane proteins. X-ray diffraction requires high quality crystals and these are not available for many membrane proteins. Circular dichroism (CD) has been widely used for studying the conformation of water-soluble proteins, but problems arise in its use for membrane proteins. The light scattering effect may distort CD spectra and lead to substantial errors in their interpretation. In addition, the reference spectra used for the analysis of CD spectra are based on globular proteins in aqueous solution and may not be applicable to membrane proteins in the hydrophobic environment of lipid bilayers. 

Induced circular dichroism (ICD) is the CD observed in an optically inactive (achiral) chromophore due to its interaction with an optically active (chiral) moiety. Early applications exploited ICD just as evidence for such an interaction. For example, the ICD in the absorption band of an achiral drug may be used to prove its binding to a (chiral) protein or nucleic acid. More recent use of ICD includes the structural information that theoretical analysis of the inducing mechanism can provide. ICD has been an important complement to linear dichroism (LD) for the assessment of binding geometries in drug-nucleic acid systems. 

---