Unraveling structural transitions and kinetics along the fold-switching pathway of the RfaH C-terminal domain using exchange-based NMR.

ABSTRACT: The bacterial transcriptional regulator RfaH comprises structurally and functionally distinct N- (NTD) and C- (CTD) terminal domains. The latter switches from a helical hairpin packed against the NTD to a five-stranded β-roll upon displacement by RNA polymerase binding. Here, we use exchange-based NMR to probe fold-switching intermediates sampled by the isolated CTD. In addition to the predominant (~76 to 77%), semistable β-roll conformation (state A), we identify four structurally and kinetically distinct states: A', B, B', and B″. State B is NMR observable with an occupancy of ~23%, exchanges slowly (τ_ex ~ 300 ms) with the major A species, and comprises a largely unfolded ensemble with transient occupancy of helical (α5*) and β-hairpin (β1*/β2*) elements. Backbone chemical shi

SUBMITTER: Cai M 

PROVIDER: S-EPMC12107155 | biostudies-literature | 2025 May

REPOSITORIES: biostudies-literature