Revealing hidden protonated conformational states in RNA dynamic ensembles

Area of Science:

• Biochemistry
• Structural Biology
• Chemical Physics

Background:

• Protonated states in RNA are crucial for biochemical reactions and pH sensing.
• Detecting short-lived, low-population protonated states is experimentally challenging.

Purpose of the Study:

• To identify and characterize a low-population protonated state in HIV-1 TAR RNA.
• To elucidate the kinetic mechanisms of protonation-coupled conformational switching in RNA.

Main Methods:

• pH-dependent NMR chemical exchange
• Kinetic solvent isotope effects
• Site-directed mutagenesis

Main Results:

• A low-populated (0.4%) conformational state of HIV-1 TAR RNA was identified, coupled to C-C mismatch protonation.
• The apparent pKa was depressed to ~4.0 due to an energetic penalty for this conformation.
• Mutation to a G-C base pair abolished pH-dependence, confirming the C-C mismatch as the protonation site.
• This hidden state competes with a C-A+ mismatch conformation, leading to non-monotonic pH response.
• Protonation-coupled switching follows an induced-fit mechanism with rapid protonation and slower structural changes.

Conclusions:

• Revealed a general mechanism for protonation-coupled conformational switching in RNA.
• Provides a framework for studying sparsely populated protonated states and their dynamics.