Development of single-molecule assays for proteolytic susceptibility: Force-induced collagen destabilization

Collagen’s unique triple helical structure allows for mechanical support and stability at all hierarchical levels including single molecules, fibrils, fibers and tissues. Surprisingly, the structural stability of the triple helix under force is not well known. I explored its response to applied force with single molecule proteolytic susceptibility assays. To perform the experiments I designed, developed and constructed a low-cost and accessible single-molecule force spectroscopy instrument called the Mini-Radio Centrifuge Force Microscope or MR.CFM. Centrifuge force microscopy allows for a wide dynamic force range, massively parallel single-molecule force measurements, and external field-free application of force. Collagen’s triple helix is resistant to proteolytic cleavage due to steric hindrance. By comparing trypsin’s cleavage rate of collagen with and without force in the MR.CFM, I monitored the triple helix’s force stability. I showed an increase of cleavage rate with force, suggesting a force-enhanced destabilization of the triple helix. One of the most challenging components of single-molecule force spectroscopy is surface chemistry; unsatisfied with available approaches I developed a surface passivation and chemical conjugation method based on an NHS end-labeled F127 Pluronic block copolymer, F127-NHS. I created an easy-to-use assay called the microsphere adhesion by gravity, inversion, then counting, or “MAGIC” assay, to quantitatively measure the interactions between a surface and microsphere. Guided by data from the MAGIC assay I reduced nonspecific interactions with a multistep labelling protocol for the microspheres. I achieved 94% of DNA microspheres singly tethered to the F127-NHS surface. In addition, I showed the surface’s utility in fluorescence and force spectroscopy assays. Taken together my work has enabled the advancement of accessible force spectroscopy and furthered the understanding of collagen’s response to force.