Knob-Socket Predictions of Alpha-Helical Stability

Abstract

Force plays a key role in regulating dynamics of biomolecular structure and interactions, yet techniques are lacking to manipulate and continuously read out this response with high throughput. We present an enzymatic assay for forcedependent accessibility of structure that makes use of a wireless Mini-Radio Centrifuge Force Microscope (MR.CFM) to provide a real-time readout of kinetics. The microscope is designed for ease of use, fits in a standard centrifuge bucket, and offers high-throughput, video-rate readout of individual proteolytic cleavage events. Proteolysis measurements on thousands of tethered collagen molecules were used to determine how the triple helix responds to force. As the primary load-and tension-bearing protein in vertebrates, the physical properties of collagen are of significant biomedical interest. How collagen's triple helix responds to applied force is controversial, with different studies inferring incompatible outcomes: overwinding, unwinding, shearing, or maintaining its zero-force structure. Because proteolytic cleavage requires a locally unwound triple helix, our experiments reveal how local collagen structure changes in response to applied force. Our first results show a load-enhanced trypsin sensitivity, indicating destabilization of the triple helix. The generality of this result will be discussed in the context of collagen's sequence heterogeneity.