Protein unfolding circular dichroism

UNFOLDED PEPTIDES AND PROTEINS STUDIED WITH INFRARED ABSORPTION AND VIBRATIONAL CIRCULAR DICHROISM SPECTRA... 

Fig. 29. Equilbrium unfolding of C40A/C82A/P27A (pseudo-wild-type) barstar monitored by A R, mean residue circular dichroism. Conditions for near-UV CD were 50 /xM protein in 50 mM Tris-HCl buffer, pH 8, 0.1 M KC1, path length 1 cm. (A) Urea-induced unfolding at 25°C at urea concentrations as indicated. (B) Cold-induced unfolding in...

Fig. 4. Time-induced conformational change of spider silk protein (spidroin) in solution. Solutions of silk proteins at 1% w/v in distilled water were monitored using circular dichroism. The graph shows a change in secondary structure with time. The silk proteins underwent a kinetically driven transition from a partially unfolded structure to a -sheet-rich structure (from Dicko et al., 2004c). ( ) after 0 days, (O) after 1 day, and (A) after 2 days. The conformational change appeared faster at 20°C compared to 5°C, suggesting a hydrophobically driven mechanism. (Copyright 2004 American Chemical Society.)...

Fig. 5. The effect of protein-protein interactions on Nephila edulis major ampullate circular dichroism spectra in solution. A change in secondary structure with increasing concentration is observed. At low concentration (minimal protein-protein interactions) silk proteins appear partially unfolded in solution. At higher concentration (higher protein-protein interactions) silk proteins refold into a helix-like structure, most likely a molten-like globule (from Dicko et al., 2004c). This final molten structure would facilitate local chain rearrangement while preserving the global structure for protein storage and transport. (Copyright 2004 American Chemical Society.)...

If the incoming light is polarised, any chiral features of the molecule that absorbs will be highlighted—in particular changes in such chiral features, e.g. when the protein is unfolding, these features will stand out. The secondary stmctural elements of proteins are chiral in their nature. This phenomenon can be followed with the so-called circular dichroism (CD). ... 

FIGURE 11.15 Resolution of the protein folding of a-apolactalbumin. (a) Detection of changes in protein secondary structure (far-UV circular dichroism measurements), (b) Detection of changes in protein tertiary structure (near-UV circular dichroism measurements), (c) Complete description of protein folding. Resolution of the row-wise data set formed by near-UV (Dj) and far-UV (D2) circular dichroism measurements. Solid line native conformation, dash-dotted line intermediate conformation, dotted line unfolded conformation. 

The nature of the unfolded state in denaturant and how it relates to the denatured state under native conditions in the bilayer is a major issue in all denaturation experiments. Thermodynamic arguments from the two-stage model suggest that the relevant denatured state has lost its tertiary structure and maintained the transmembrane helix secondary structure. As noted above, CD spectra on thermally denatured bacteriorhodopsin suggest that the denatured protein maintains most of its helical secondary structure. The extent to which tertiary structure is disrupted is unclear, however. It is possible that some stable interhelical interactions are maintained even at high temperature. The helical secondary structure content is also maintained in SDS micelles, and near-UV circular dichroism (CD) spectra suggest substantial loss or... 

For comparative purposes, the stability of a protein needs to be referred to specific standard conditions this is usually 25 °C and aqueous solution, and is denoted by the symbols A G25 and A G ,z0 respectively. In order to determine the values of A G° parameters, observations must be made of some intrinsic parameter that changes when the protein unfolds. This can be UV absorption, fluorescence, optical rotary dispersion (ORD) or circular dichroism (CD) (Sect. 7.1), the latter being particularly useful since it can be used to establish when the unfolding process is complete. This is because the CD spectra of fully unfolded proteins are very similar to one another, and very different from those of native proteins which contain a-helices and /3-pleated sheet structures (Figure 5-12). 

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