Using DNA as a fiducial marker to study SMC complex interactions with the atomic force microscope.

Area of Science:

• Biophysics
• Structural Biology
• Microscopy

Background:

• Atomic force microscopy (AFM) offers insights into protein characteristics but is prone to tip-induced artifacts.
• Accurate measurement of protein volumes, shapes, and interactions is crucial for understanding protein function.

Purpose of the Study:

• To develop a novel AFM approach for precise volume measurements of nonglobular proteins, specifically structural maintenance of chromosomes (SMC) proteins.
• To investigate the interactions and oligomeric states within and between Bacillus subtilis SMC complexes.

Main Methods:

• Co-adsorption of DNA molecules with target proteins on AFM substrates.
• Utilizing DNA as a fiducial marker to normalize protein volumes and correct for tip-induced artifacts.
• AFM imaging to determine oligomeric states and architecture of SMC complexes.

Main Results:

• The developed AFM method significantly reduced volume analysis errors.
• Determined the oligomeric states: SMC and ScpB are dimers, ScpA is a monomer.
• Elucidated interaction pathways: ScpA binds directly to SMC; ScpB binds to SMC only when ScpA is present.

Conclusions:

• The novel AFM approach enables accurate volume quantification of challenging protein samples.
• The study reveals the subunit composition and architecture of the Bacillus subtilis SMC complex.
• ScpA and ScpB play critical roles in organizing higher-order SMC complex structures.