Imaging and three-dimensional reconstruction of chemical groups inside a protein complex using atomic force...

Area of Science:

• Biophysics
• Structural Biology
• Nanotechnology

Background:

• Scanning probe microscopy (SPM) excels at atomic-scale surface characterization.
• SPM's tip-sample interactions limit imaging to the topmost atomic layer.
• Characterizing internal structures of biomolecules remains a challenge for conventional SPM.

Purpose of the Study:

• To demonstrate atomic force microscopy (AFM) for 3D imaging and reconstruction of internal chemical groups in protein complexes.
• To overcome the surface-limitation of traditional SPM for internal biomolecular structure determination.

Main Methods:

• Utilized short single-stranded DNA (ssDNA) as sequence-specific imaging labels attached to target protein regions.
• Employed T-shaped AFM cantilevers functionalized with complementary DNA probes for label localization.
• Measured pairwise distances between labeled sites for 3D reconstruction via geometric calculations.

Main Results:

• Successfully imaged and 3D reconstructed chemical groups within a protein complex.
• Achieved subnanometer resolution and sequence specificity in label localization.
• Demonstrated high accuracy, with biotin carboxylic acid group loci within 2 Å of crystal structure data in biotin-streptavidin complex experiments.

Conclusions:

• AFM, with DNA labeling and complementary probes, can visualize and reconstruct internal chemical group structures in proteins.
• This method offers a powerful complement to existing structural biology techniques for large and complex biomolecules.
• Paves the way for detailed structural analysis of previously inaccessible internal biomolecular features.