Isothermal titration calorimetry as a method to assess the binding kinetics of Syntaxin4, SNAP23, VAMP2 and Munc18c

Q Science (General)

Fuller, Andrew ; Gould, Gwyn

University:University of Glasgow

Department:Institute of Molecular Cell and Systems Biology

关键词: GLUT4, SNARE, Syntaxin 4, SNAP23, VAMP2, Munc18c, isothermal titration calorimetry;   

Others  :  http://theses.gla.ac.uk/4087/1/2013FullerMSc.pdf

来源: University of Glasgow

【 摘 要 】

The ability of humans to regulate their blood glucose using insulin responsive translocation of the glucose transporter GLUT4 to the plasma membrane is key to survival. In the absence of insulin GLUT4 is stored within intracellular vesicles which upon insulin stimulation translocate to the plasma membrane and then fuse with the plasma membrane. This fusion step is driven by a group of proteins known as SNARE proteins more specifically syntaxin 4 and SNAP23 in the plasma membrane and VAMP2 in the vesicle. Located in the plasma membrane and the intracellular vesicles, SNARE proteins are able to form a highly stable complex through their SNARE domains. The binding of SNARE domains to one another brings the vesicle and plasma membrane within close proximity, as the SNARE proteins form a stable complex the energy released is theorised to be sufficient to drive fusion of the vesicle and plasma membrane. The formation of the SNARE complex is regulated by the isolation of the contributing SNARE proteins and by other proteins which can bind both individual SNARE proteins and the SNARE complex as a whole. Munc18c is a member of one such class of proteins, Sec/Munc (SM) proteins and has been shown to bind to syntaxin 4 and the complete SNARE complex of syntaxin 4, SNAP23 and VAMP2. The exact amount of energy released upon the stable binding of SNARE domains in SNARE proteins and their associated regulatory proteins such as SM proteins is still a debated subject. This project aims to elucidate this information by utilising the highly sensitive technique isothermal titration calorimetry and recombinant protein synthesis and purification of the SNARE proteins syntaxin 4, SNAP23 and VAMP2 and the SM protein Munc18c.