Folding/unfolding/refolding of proteins: Present methodologies in comparison with capillary zone electrophoresis

A series of techniques for monitoring protein folding/unfolding/misfolding equilibria are here assessed and compared with capillary zone electrophores is (CZE). They include spectroscopic techniques, such as circular dichroism , intrinsic fluorescence, nuclear magnetic resonance, Fourier transform inf rared and Raman spectroscopy, small-angle X-ray scattering, as well as tech niques based on biological assays, such as limited proteolysis and immunoch emical analysis of different conformational states. Some unusual probes, su ch as mass spectrometry for probing unfolding transitions, are also discuss ed. Size-exclusion chromatography is also evaluated in view of the fact tha t this technique, like all electrophoretic techniques, and unlike spectrosc opic probes, which can only see an average signal in mixed populations, can indeed physically separate folded vs. unfolded macromolecules, especially in the case of slow equilibria. Particular emphasis is devoted to electroph oretic techniques, such as gel-slab electrophoresis in transverse urea or t hermal gradients, and CZE. In the latter case, a number of applications are shown, demonstrating the excellent correlation of CZE with more traditiona l probes, such as intrinsic fluorescence monitoring. It is additionally sho wn that CZE can be used for measuring the DeltaG degrees of unfolding over the pH scale, in good agreement with theoretical calculations on the electr ostatic free energy of folding vs. pH, as calculated with a linearized Pois son-Boltzmann equation. Finally, it is demonstrated that CZE can probe also aggregate formation in the presence of helix-inducing agents, such as trif luorethanol.