Deprotonation stimulates productive folding in allosteric TRAP hammerhead ribozymes

Hammerhead ribozymes in crystals change conformation in response to deprotonation of the nucleophilic 2' OH, thereby aligning the hydroxyl for in-line displacement at the scissile phosphate. Published data do not address whether deprotonation affects folding in solution. Allosteric hammerhead "TRAPs," when activated by the appropriate oligonucleotide, show the expected log-linear relation between initial cleavage rate and pH. In contrast, attenuated TRAPs shows biphasic kinetics in which a rapid burst is followed by slow cleavage that is nearly independent of pH. Attenuated ribozymes are stimulated by urea at both low and high pH, confirming that rearrangement of secondary structure is rate-limiting for the attenuated ribozymes once they have folded. Plots of burst magnitude versus pH in the absence of urea show a sharp transition around pH 8.3, which is near the kinetic pKa for the cleavage reaction in Mg2+. Raising the pH after folding at pH 7.5 did not activate attenuated ribozymes even when the RNA was incubated at the elevated pH for extended periods prior to addition of Mg2+. In contrast, lowering the pH after folding at pH 9.5 rapidly re-established attenuation. Deprotonation of the ribozyme-substrate complex thus appears to alter the folding landscape such that a metastable "pre-activated" complex forms before the thermodynamically more stable attenuated state can be attained. From the initial partition into active and inactive conformers, we estimate that this deprotonation contributes approximately 1.2 kcal/mol toward stabilization of the active fold at a crucial step during folding of the TRAP. Assuming that the nucleophilic 2' OH is the relevant acid, its deprotonation would thus serve a dual role of favoring productive fold and enhancing the nucleophilicity of this oxygen.