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Description:
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Synaptotagmin I is the most abundant Ca+2 binding protein present on synaptic vesicles accounting for 7 % of total vesicle protein and is widely accepted as the Ca2+ sensor in fast synchronous neurotransmitter release . The protein is composed of one trans -membrane domain , an unstructured linker followed by two C2A domains identified as C2A and C2B . Each C2 domain is composed of an 8 stranded β -sandwich joined by a 9 amino acid linker . The Ca+2 binding pocket is composed of three loops located at the apex of the protein . In the Syt I C2AB structure , we see evidence of a domain structural change in the absence of Ca2+ . Analysis of interacting residues between C2A and C2B show a network of highly conserved residues within the C2 domain that regulates Ca+2 /phohspholipid binding in C2A . Analysis of the Syt I C2A structure , as well as , previous C2A structures shows a strong H -bond between Tyr 180 and His 237 in C2A . By removing this H -bond , disorder of Loop 3 is increased and the thermodynamic stability of the C2 domain decreases . Our hypothesis is that the absolute position of the Ca2+ binding loops of C2 domains affects Ca+2 affinity and , and ultimately domain stability . We used several different biochemical approaches to test the hypothesis . We assessed the importance of Loop 3 mutations using X -ray crystallography methods , bulk thermodynamic measurements using lifetime fluorescence , and analyzed the mechanical properties of the C2 -domains using single molecule force spectroscopy .
We studied the mechanical stability of the C2A and C2B domains of human Syt1 using single -molecule atomic force microscopy . We found that stretching the C2AB domains of Syt1 resulted in two distinct unfolding force peaks . The larger force peak of ~100pN was identified as C2B and the second peak of ~50pN as C2A . Further , a significant fraction of C2A domains unfolded through a low force intermediate that was not observed in C2B . We conclude that these domains have different mechanical properties . We hypothesize that a relatively small stretching force may be sufficient to deform the effector -binding regions of C2A domain and modulate the affinity for SNAREs , phospholipids and Ca+2 . |