Diverse single-stranded nucleic acid binding proteins enable both stable protection and rapid exchange required for...

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Single-stranded nucleic acid (ssNA) binding proteins perform dual roles: protecting exposed ssNA during replication and facilitating its processing.
• The coexistence of these opposing functions in ssNA binding proteins has been a key question in molecular biology.

Purpose of the Study:

• To elucidate the mechanisms by which ssNA binding proteins achieve both stable protection and rapid reorganization of ssNA.
• To compare single-molecule experimental data from diverse proteins exhibiting similar ssNA interactions.

Main Methods:

• Utilized optical tweezers (OT) to isolate and manipulate single long ssNA molecules.
• Measured ssNA conformation changes in real-time upon protein binding to quantify interaction structure and kinetics.
• Compared OT data from three distinct ssNA binding proteins.

Main Results:

• OT experiments revealed that protein binding alters the effective length of ssNA substrates.
• Observed complex OT signals, including non-monotonic responses to protein concentration, indicating multiple binding states.
• Identified a general model where protein oligomers switch conformations to modulate protein:NA stoichiometry.

Conclusions:

• Protein oligomers with multiple binding interfaces can adjust their conformation to regulate protein:NA stoichiometry.
• This conformational flexibility allows proteins to maximize contacts for ssNA protection while enabling efficient reorganization and displacement.
• The findings provide a generalizable model for understanding ssNA binding protein function across different protein families.