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

Abstract

The folding kinetics of the multidomain ribosomal protein L9 were studied using pH jump stopped-flow fluorescence and circular dichroism (CD) in conjunction with guanidine hydrochloride (GdnHCl) jump stopped-flow CD experiments. Equilibrium CD and 1D (1)H NMR measurements demonstrated that the C-terminal domain unfolds below pH 4 while the N-terminal domain remains fully folded. Thus, the N-terminal domain remains folded during the pH jump experiments. 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 measurements showed that the N- and C-terminal domains fold independently each by an apparent two-state mechanism. The folding rate constant for the N-terminal domain and the C-terminal domain in the absence of denaturant were calculated 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 folding 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-terminal domain is not a consequence of unfavorable interactions with the rest of the protein chain during refolding. This is an interesting result since contact order analysis predicts that the folding rate of the C-terminal domain 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 as the rate for the domain in the intact protein.

Early Collapse is not an Obligate Step in Protein Folding

“…For ctAcP, the change in CD is fully accounted for by a sloping base-line of the unfolded state. All the stopped-flow CD data on kinetically two-state proteins we are aware of share this property 41,42,78 although the base line sometimes has mild curvature. 44,46,79 For Ub, the burst-phase CD has pronounced increase starting at 2.5 M GdmCl.…”

Analysis of the pH-dependent Folding and Stability of Histidine Point Mutants Allows Characterization of the Denatured State and Transition State for Protein Folding

“…[20][21][22] CTL9 contains 92 residues (residues 58-149 of the ribosomal protein L9) and consists of a threestranded b-sheet with an unusual mixed parallel and anti-parallel architecture ( Figure 1). The rare topology leads to a slow folding rate, [23][24][25] and makes CTL9 an interesting target for folding studies. CTL9 contains three histidine residues, H144, H134 and H106.…”

Fully Reduced Ribonuclease A Does not Expand at High Denaturant Concentration or Temperature

“…40 Nevertheless, submillisecond "burst-phase" circular dichroism (CD), fluorescence and fluorescence resonance energy transfer (FRET) signals have been observed for many twostate proteins. 18,29,30,[40][41][42][43][44][45][46][47] We have proposed that these signal changes often represent the adjustment of the denatured state to the new, poorer solvent condition according to the unfolded state baseline, rather than the formation of a distinct intermediate. 40,44,[48][49][50] This view remains controversial, 10 particularly for RNase A.…”