High-resolution atomic force microscopy visualization of metalloproteins and their complexes

Area of Science:

• Biochemistry
• Biophysics
• Molecular Biology

Background:

• Metalloproteins myeloperoxidase (MPO), ceruloplasmin (CP), and lactoferrin (LF) are key regulators of inflammation and oxidative stress.
• Previous research indicated synergistic antimicrobial and anti-inflammatory roles of MPO-CP and LF-CP complexes.
• Single-molecule interactions within these metalloprotein complexes remained uncharacterized.

Purpose of the Study:

• To investigate pairwise interactions of MPO, CP, and LF molecules at the single-molecule level.
• To characterize the morphology of MPO-CP and LF-CP complexes.
• To develop a reliable method for AFM investigation of metalloproteins.

Main Methods:

• High-resolution atomic force microscopy (AFM) was employed.
• Proteins were deposited on a highly oriented pyrolytic graphite (HOPG) surface modified with oligoglycine-hydrocarbon graphite modifier (GM).
• Single metalloprotein molecules and their complexes were visualized and analyzed.

Main Results:

• A novel procedure for reliable AFM investigation of metalloproteins was established.
• Single MPO, CP, and LF molecules were visualized for the first time.
• The morphology of MPO-CP and LF-CP complexes was characterized, revealing a chainlike structure for MPO-CP conjugates and confirming the absence of direct MPO-LF contacts.

Conclusions:

• GM-HOPG serves as an effective substrate for AFM studies of metalloproteins.
• AFM visualization provides new insights into the ultrastructure of MPO-CP complexes, complementing bulk techniques.
• The findings enhance understanding of inflammation and oxidative stress regulation through metalloprotein collaboration.