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 electrophoresis (CZE). They include spectroscopic techniques, such as circular dichroism, intrinsic fluorescence, nuclear magnetic resonance, Fourier transform infrared and Raman spectroscopy, small-angle X-ray scattering, as well as techniques based on biological assays, such as limited proteolysis and immunochemical analysis of different conformational states. Some unusual probes, such as mass spectrometry for probing unfolding transitions, are also discussed. Size-exclusion chromatography is also evaluated in view of the fact that this technique, like all electrophoretic techniques, and unlike spectroscopic 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 electrophoretic techniques, such as gel-slab electrophoresis in transverse urea or thermal gradients, and CZE. In the latter case, a number of applications are shown, demonstrating the excellent correlation of CZE with more traditional probes, such as intrinsic fluorescence monitoring. It is additionally shown that CZE can be used for measuring the deltaG degrees of unfolding over the pH scale, in good agreement with theoretical calculations on the electrostatic free energy of folding vs. pH, as calculated with a linearized Poisson-Boltzmann equation. Finally, it is demonstrated that CZE can probe also aggregate formation in the presence of helix-inducing agents, such as trifluorethanol.
Righetti
P G
PG
University of Verona, Department of Agricultural and Industrial Biotechnologies, Italy. righetti@mailserver.unimi.it
Verzola
B
B
D017766:Acid Anhydride Hydrolases / Q000737:chemistry; D002151:Calorimetry; D002850:Chromatography, Gel; D002942:Circular Dichroism; D019075:Electrophoresis, Capillary; D010450:Endopeptidases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007118:Immunoassay; D013058:Mass Spectrometry; D008832:Microchemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman; D014961:X-Ray Diffraction; D000097024:Acylphosphatase
D017766:Acid Anhydride Hydrolases; D010450:Endopeptidases; D010446:Peptide Fragments; D000097024:Acylphosphatase