pH jump studies of the folding of the multidomain ribosomal protein L9: The structural organization of the N-terminal domain does not affect the anomalously slow folding of the C-terminal domain

The folding kinetics of the multidomain ribosomal protein L9 were studied u sing pH jump stopped-flow fluorescence and circular dichroism (CD) in conju nction with guanidine hydrochloride (GdnHCl) jump stopped-flow CD experimen ts. Equilibrium CD and 1D H-1 NMR measurements demonstrated that the C-term inal domain unfolds below pH 4 while the N-terminal domain remains fully fo lded. Thus, the N-terminal domain remains folded during the pH jump experim ents. The folding rate constant of the C-terminal domain was determined to be 3.5 s(-1) by pH jump experiments conducted in the absence of denaturant using stopped-flow CD and fluorescence. CD-detected GdnHCl jump measurement s showed that the N- and C-terminal domains fold independently each by an a pparent two-state mechanism. The folding rate constant for the N-terminal d omain and the C-terminal domain in the absence of denaturant were calculate d to be 760 and 4.7 s(-1), respectively. The good agreement between the pH jump and the denaturant concentration jump experiments shows that the foldi ng rate of the C-terminal domain is the same whether or not the N-terminal domain is folded. This result suggests that the slow folding of the C-termi nal domain is not a consequence of unfavorable interactions with the rest o f the protein chain during refolding, This is an interesting result since c ontact order analysis predicts that the folding rate of the C-terminal doma in should be noticeably faster. The folding rate of the isolated N-terminal domain was also measured by stopped-flow CD and was found to be the same a s the rate for the domain in the intact protein.