Two molecule force spectroscopy on ligand-receptor interactions.

Abstract

Many biological processes involve the rupture of multiple ligand-receptors or multivalent ligand-receptors. It is challenging to study the rupture of such parallelly arranged multiple ligand-receptors due to the difficulties in engineering such systems in a well-controlled fashion. Here we report the use of two-molecule force spectroscopy to investigate the rupture of two parallelly arranged monomeric streptavidin (mSA)-biotin complexes. By using SpyCatcher-SpyTag chemistry, we successfully engineered a molecular twin of biotin, in which two biotins are arranged in parallel. By reacting mSA with twin biotin, we constructed parallelly arranged two mSA-biotin complexes for force spectroscopy experiments. The incorporation of single molecule fingerprint domains into our mSA-biotin dimers allowed us to identify and assign the rupture events of the parallelly arranged mSA-biotin complexes without any ambiguity in the two-molecule force spectroscopy experiments. Our results revealed that the rupture force of the parallel dimer mSA-biotin is 172 pN at a pulling speed of 400 nm s-1, which is about 1.6 times of that of single mSA-biotin (105 pN). Furthermore, our findings indicate that the two mSA-biotin behave as non-interacting, independent ligand-receptors. The strategy we demonstrated here can be extended to other ligand-receptors and may open up an avenue toward rigorously testing the theoretic predictions proposed in various models regarding the rupture of multiple parallel ligand-receptors.