Monitors of conformational changes

SEC has its place in the purification and isolation of biopolymers, e.g. insulin, and other therapeutic drugs as a module in downstream processing [8, 9]. Mass spectrometry has, however, comprehensively displaced S EC for molecular weight determination. A current field of application is in the monitoring of conformational changes and aggregation of proteins. Micro-SEC with capillary columns has foimd application in the SEC of synthetic polymers in process analysis. 

Despite its weakness, the anisotropy of the g tensor of iron-sulfur centers can be used to determine the orientation of these centers or that of the accommodating polypeptide in relation to a more complex system such as a membrane-bound complex. For this purpose, the EPR study has to be carried out on either partially or fully oriented systems (oriented membranes or monocrystals, respectively). Lastly, the sensitivity of the EPR spectra of iron-sulfur centers to structural changes can be utilized to monitor the conformational changes induced in the protein by different factors, such as the pH and the ionic strength of the solvent or the binding of substrates and inhibitors. We return to the latter point in Section IV. 

The application of heterobifunctional cross-linkers allows macromolecular PAL to probe protein-protein interactions, including subunit interactions and location, monitoring the conformational changes induced by signal transmission. 

The simplicity of performing this protein secondary structure analysis makes CD spectroscopy useM in monitoring protein conformational changes between wild-type and mutant proteins, as well as in identifying changes in the protein secondary structure upon addition of a bound cofactor such as a metal ion. Several recent examples of applying CD spectroscopy for these purposes are now described. 

Equilibrium Denaturation. A variety of different techniques can be employed to monitor protein conformational changes in the presence of denaturants. Activity measurements reflect the extent of alterations of the active site environment. However, enzyme activity measurements may be affected the presence of denaturant in the assay mixture. The denaturation curves obtained by this method are difficult to inte ret and can only be taken as a first approximation of the unfolding transition. U.V. difference spectra indicate conformational changes by monitoring the degree of solvent exposure of aromatic amino-acid side chains. Finally, fluorescence intensity measurements can reveal the nature of the environment (polar, non-polar) of the four tryptophans of p-lactamase. 

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