MONITORING THE REFOLDING PATHWAY FOR A LARGE MULTIMERIC PROTEIN USINGCAPILLARY ZONE ELECTROPHORESIS

Rapid identification of transient partially folded intermediates forme d during protein refolding and aggregation has been difficult, particu larly with separation methods relying on solid matrices. Capillary zon e electrophoresis equipped with laser-induced fluorescence detection p rovides a fast sensitive means of identifying folding and aggregation intermediates using the intrinsic tryptophan fluorescence. The in vitr o refolding of the trimeric P22 tailspike, a model system for the stud y of protein folding, misfolding and aggregation, has been monitored a fter dilution out of denaturant. Both monomeric and trimeric folding i ntermediates were resolved. The refolding kinetics and yields measured by capillary zone electrophoresis were in good agreement with those o btained via fluorescence spectrophotometry and polyacrylamide gel elec trophoresis. In comparison with typical UV detection, laser-induced tr yptophan fluorescence increased detection sensitivity. In addition, th e fluorescence signal carries information on the packing of the trypto phan residues in the folding intermediates. For tailspike and many oth er proteins, the off pathway aggregation reactions proceed from a ther molabile intermediate at the junction with the productive pathway. By monitoring refolding intermediates after temperature shifts, the struc tured monomeric intermediate was identified as the thermolabile juncti onal intermediate between the productive and aggregation pathways.