Quantifying the Relationship between Single-Molecule Probes and Subunit Rotation in the Ribosome

Area of Science:

• Biophysics
• Molecular Biology
• Structural Biology

Background:

• Interpreting single-molecule fluorescence resonance energy transfer (smFRET) measurements requires precise reaction coordinates for biomolecular assemblies.
• Conflicting descriptions of ribosome subunit rotation during mRNA-tRNA translocation exist, stemming from different experimental probes and labeling strategies.

Purpose of the Study:

• To rationalize conflicting single-molecule measurements of ribosome subunit rotation.
• To develop a physically grounded framework for understanding subunit rotation and its coupling to translocation.

Main Methods:

• Simulated hundreds of spontaneous ribosome subunit rotation events (≈8°) using a residue-level coarse-grained model.
• Analyzed nine different reaction coordinates to assess their ability to monitor conformational changes.

Main Results:

• Found that apparent inconsistencies in experimental measurements are likely due to inherent ribosomal flexibility.
• Proposed a novel metric to quantify the energetic coupling between experimentally measured degrees of freedom and subunit rotation.

Conclusions:

• Ribosomal flexibility is a key factor in interpreting smFRET data related to subunit rotation.
• The proposed framework and metric can guide the development of more precise single-molecule techniques for studying biomolecular dynamics.