ABSTRACT
Fan, Z.H., Jensen, P.K., King, J. and Lee, C.S. (1997)
J. Chromatography, 769,
315-323
Monitoring the refolding pathway for a large multimeric protein using capillary zone electrophoresis
Rapid identification of transient partially folded intermediates formed during protein refolding and aggregation has been difficult, particularly with separation methods relying on solid matrices. Capillary zone electrophoresis equipped with laser-induced fluorescence detection provides a fast sensitive means of identifying folding and aggregation intermediates using the intrinsic tryptophan fluorescence. The in vitro refolding of the trimeric P22 tailspike, a model system for the study of protein folding, misfolding and aggregation, has been monitored after dilution out of denaturant. Both monomeric and trimeric folding intermediates were resolved. The refolding kinetics and yields measured by capillary zone electrophoresis were in good agreement with those obtained via fluorescence spectrophotometry and polyacrylamide gel electrophoresis. In comparison with typical UV detection, laser-induced tryptophan fluorescence increased detection sensitivity. In addition, the fluorescence signal carries information on the packing of the tryptophan residues in the folding intermediates. For tailspike and many other proteins, the off pathway aggregation reactions proceed from a thermolabile intermediate at the junction with the productive pathway. By monitoring refolding intermediates after temperature shifts, the structured monomeric intermediate was identified as the thermolabile junctional intermediate between the productive and aggregation pathways.
