Development of glutathione-coupled cantilever for the single-molecule force measurement by scanning force microscopy

Abstract 

The accuracy and the fidelity of a single-molecule force measurement largely rely on how the molecule of interest is attached to the solid substrate surface (bead, cantilever, cover glass and etc.). A site-specific attachment of a protein without affecting its structure and enzymatic function has been a major concern. Here, we established a glutathione-coupled cantilever to which any glutathione S-transferase (GST)-fused proteins can be attached in a desired direction. The rupture force between glutathione and GST was ∼100pN on average. By using this cantilever, we succeeded in measuring the interaction force between importin α and importin β.

Abbreviations: AFM, atomic force microscope, APTES, 3-aminopropyltriethoxysilane, SATP, N-succinimidyl S-acetylthiopropionate, PBS, phosphate buffered saline, NHS, N-hydroxysuccinimide, GST, glutathione S-transferase, DTT, dithiothreitol

Keywords: Scanning probe microscope, Single-molecule force measurement, Cantilever modification, Importin α, Importin β, Nuclear transport