Entschlüsselung des Einflusses von in spondyloepiphysärer Dysplasie vorkommender struktureller Veränderungen auf die mechanischen Eigenschaften von Kollagen Typ II
Zusammenfassung der Projektergebnisse
Collagen provides elasticity and support to cells and tissues. Its unique triple helical structure is thought to impart mechanical stability under a wide range of forces. Until today, the influence of force on its molecular mechanics is not well understood. This project aimed at answering the central question of how an applied force affects the quaternary structure of collagen. By unraveling the mechanical properties of wild-type collagen I planned to determine structural and mechanical aberrations of a series of type II collagen mutations to elucidate how mutations may change collagens’ behaviour under force. For preliminary Centrifuge Force Microscopy (CFM) experiments Michael Kirkness, a PhD student in Nancy Forde’s lab, had already been able to specifically tether single collagen molecules and demonstrated the feasibility for cleavage by different enzymes, under force, and at different temperatures. He revealed that trypsin cleavage of collagen is facilitated by applied forces of 10 pN at room temperature. Due to steric hindrance trypsin cleavage cannot occur in the intact triple helix, hence collagen appears to be unwound under those experimental conditions. However, the fraction of beads remaining on the surface was always found to be around 50% at the end of the experiment. Hence, the beads appeared to be non-specifically bound to the surface. Aim1: Improvement of surface chemistry. By using the polymeric F127F127-Hydrazide I was able to decrease the amount of non-specifically bound beads to ~5%. Aim2: Improvement and up-scaling of chamber preparation. By designing a glass cleaning devise (Figure 5) I was able to up-scale and significantly accelerate the cleaning procedure. The device consists of three glass layers which can each hold 22 customized cover slips or glass slides. The layers can be staggered on top of each other and then fully emerged into a beaker filled with a liquid of choice. This significantly improve the entire glass cleaning protocol which is now routinely used by all members of Nancy Forde’s Lab. Aim3: CFM experiments wt and mutated Collagen. Unfortunately, I was not able to reproduce the results obtained by Mike Kirkness. In my CFM experiments there was no detectable difference between the untreated and the Trypsin treated collagen sample under force. To verify that the beads were specifically tethered via collagen to the glass surface I performed two independent assays: 1) MAGIC assay with Collagenase: led to the expected decrease in amount of beads bound to the surface. Hence, the beads appeared to be specifically bound to the surface via collagen. 2) SDS-Gel analysis of Trypsin activity: untreated and preheated collagen (60 °C, 20min) incubated with and without Trypsin were compared; Trypsin activity was validated as the amount of full-length Collagen was significantly decreased in the preheated (destabilized) sample. Unfortunately, “tree like” structures were visible with AFM imaging, which indicates that the collagen tethers on the bead surface consist of more than one collagen molecule. Hence, in the CFM experiments Trypsin cleavage might be hindered and the applied force would be lower than the calculated 10 pN. In both cases Collagen destabilization and Trypsin cleavage might be impaired, leading to a plausible explanation for the absence of cleavage events. We are currently trying to circumvent this by decreasing the collagen concentration during the Aldehyde functionalization step. Due to the current situation in the lab (COVID-19 pandemic) no recent results can be shown here.
Projektbezogene Publikationen (Auswahl)
- Mechanics and Structural Stability of the Collagen Triple Helix. Current Opinion in Chemical Biology 53, 98-105 (2019)
M.W.H. Kirkness, K. Lehmann and N.R. Forde
(Siehe online unter https://doi.org/10.1016/j.cbpa.2019.08.001) - Optical tweezers approaches for probing multiscale protein mechanics and assembly. Frontiers in Molecular Biosciences, section Nanobiotechnology
K. Lehmann, M. Shayegan, G. A. Blab, N. R. Forde
(Siehe online unter https://doi.org/10.3389/fmolb.2020.577314)
