I'm looking into the possibility of building a construct containing one of
the C2 domains from a large protein and overexpressing it in a bacterial
system. However I'm a complete novice in this game (I'm a third year medical
science undergraduate working part-time in a diagnostic lab).
Briefly, C2 domains consist of a sandwich of two antiparallel beta sheets,
each of which contains four strands. Loops at one end of the sandwich form a
calcium-binding region and can also be involved in calcium-dependent binding
to proteins or phospholipids. C2 domains from a number of proteins (e.g.
protein kinase C, cytosolic phospholipase A2, a number of synaptotagmins)
fold independently and have been successfully expressed in bacteria.
I've identified the core of the domain in this particular protein, but I'm
having trouble finding out where the domain begins and ends. The problem is
that the domains can have either of two different topologies, in which seven
beta strands are conserved but the eighth can be at either the N-terminal or
the C-terminal end of the primary sequence. Both topologies produce
essentially the same basic tertiary structure, except that one has the N-
and C-termini at the calcium-binding end of the domain while the other has
these termini at the opposite end. I'm not sure which topology my protein
has, and I'm finding it difficult to figure out how to find out.
I guess what I'd love is for some expert on C2 domains to turn up and tell
me exactly what I need to know, but in the absence of such an expert any
advice or suggestions would be greatly appreciated. :-)