Unravelling the mechanical properties of structural aberration of type II collagen causing Spondyloepiphyseal dysplasia

• Applicant: Dr. Kathrin Lehmann
• Subject Area: Biophysics
• Term: from 2018 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 415037474

Collagen, one of the most abundant proteins in vertebrates, plays a crucial role for the integrity of various tissues. All collagen types consist of three α chains, which are coiled about each other into a right-handed triple helix and self-assemble into higher order structures. As collagen is the major component of cartilage, which supports load and guarantees frictionless motion between joint surfaces, it is surprising that collagens’ mechanical properties under force have not yet been fully understood. Hitherto, contradicting hypotheses have been proposed: 1) overwinding or 2) underwinding of the triple helical structure under force. All force-dependent collagen experiments at the molecular level have employed magnetic or optical tweezers, costly techniques that are generally low-throughput and require substantial technical expertise. A newly developed technique – centrifuge force microscopy (CFM) helps to surmount these issues. CFM as a high-throughput single-molecule stretching instrument enables force-dependent structural studies with temperature control, live video, long run times (> 2 hours) and a low build cost (< $1000). The system consists of a miniature light microscope mounted within a rotating device like a centrifuge bucket, thus enabling single molecule measurements on an ensemble of objects using centrifugal force. This technique elegantly allows force-dependent measurements in a high-throughput manner. Changes of collagen’s triple helical structure under force will be detected by a carefully selected variety of enzymes, which either preferentially cut the intact or unwound triple helix. I will be able to analyze local and global changes of collagen’s structure under various forces and temperatures to characterize the mechanical properties of wild type type II collagenThis knowledge will then be used to analyze mechanical aberrations caused by mutations leading to single amino acid exchanges, which have been reported to be associated with Spondyloepiphyseal dysplasia (SED). SED describes a heterogeneous group of hereditary skeletal disorders affecting both normal growth and accurate remodelling of cartilage and bones. With the already established recombinant expression system I will be able to understand how prevalent mutations found in SED patients may change collagen’s mechanical properties and how applied forces affect its quaternary structure in vitro. My aim is to investigate the interplay of force and temperature in respect to the triple helical structure of wild type and mutated type II collagen, known to be involved in SED, by performing force and temperature-dependent enzymatic cleavage kinetic using CFM. The thereby obtained results could become the coveted last clue to solving the mystery of collagens’ genotype - phenotype association and might help to explain the in vivo occurring symptoms of SED associated with single amino acid exchanges from a mechanical point of view.

• DFG Programme: Research Fellowships
• International Connection: Canada
• Host: Professorin Nancy Forde