Endothelial cells sense laminar fluid shear stress for stabilization, whereas sensing disturbed shear triggers local inflammation that leads to myocardial infarction, ischemic stroke or peripheral artery diseases, which are major killers in developed countries. Mechanotrasduction events are cellular signaling responses to biophysical forces. Many studies have reported a litany of molecules involved in mechanotransduction. Schwartz lab has been identified mechano-sensitive proteins that are thought to be activated as a direct result of force. Among them, I am particularly interested in PECAM-1(PECAM), as it plays an important role in the pro- vs. anti- inflammatory responses to shear stress described above. Though many studies are implicating PECAM as a true mechano-transducer, how force activates PECAM is poorly understood. I hypothesize that particular domains of the PECAM extracellular part are activated by force. But, unfortunately existing structural methods are not compatible with structural analysis of proteins in situ under force to answer this question. To solve this problem, here I propose to develop a DNA origami device for applying defined forces to proteins, which I will combine with state of the art cryo electron microscopy (cryoEM) to determine protein structure under force. To accomplish this goal, I have done rigorous proof of concept experiments. I modified previously reported V-shaped DNA origami device, and successfully attached proteins on it. I can adjust the angle of the device to apply range of force to the protein. CryoEM imaging condition of the device has been optimized. Finally, I have purified 0.8mg of PECAM extracellular domain, which is enough for further experiments. Based on these results, I propose following specific aims. 1) I will further calibrate the DNA origami nanodevice to precisely regulate applied force to the connected proteins in the range of 1-10 pN. 2) I will obtain structure of PECAM at different force magnitudes with cryoEM. The resulting protein structure under tension will stimulate many flow-up projects including mutagenesis study in the cells and generating knock in mouse models containing mutations at force-sensitive domains. Notably, the same approach can be applied to many other mechano-sensitive proteins. In short, a breakthrough method to study force-induced conformational changes of proteins will be developed through this project, which is to find wide utility across many fields in biology.
|Program type||Postdoctoral Fellowship|
|Effective start/end date||01/01/2020 → 12/31/2021|