I'm thinking of crosslinking two proteins. The crystallographic nature of the interaction and the binding motif for each molecule is know. The paper says the intermolecular contacts (over 50; van der Waals radius 5 angstrom) are made between the motifs in these two proteins. I also know that one of the motifs on one protein has a sulfhyryl (Methionine) and for the other motif on the other molecule I (think) can use a primary amide reactive moieties. Now this is where my question is.
Would I be correct in thinking that the best cross linker to use is a heterobifunctional cross linker, with a spacer of 10 (or 5?) angstrom in order to pick this interaction up specifically and stabilise it?
I desperately need a guidance on this. I have used the life technologies crosslinker selection tool and the crosslinking technologies hand book but I can't quite figure out based on the information I have, what cross linker length I need to use. I'm not worried about the cleavability, solubility and membrane permeability parameters.
I know that the binding motif on the first protein it has Tyrosine, Proline and Methionine.
Looking at the groove contacts, the second protein is interacting with this motif, using Tyrosine and Glutamine BUT the second protein also has Tryptophan, Lysine, Glutamic acid and Methionine.
I found specially close Glutamic acid (E) and leucine (L) on my first protein, specially close to Lysine (L) on my second protein although its not in the consensus sequence for the second protein. They are less than 10 angstrom apart. is that any better? So presumably I can use the homobifunctional linker?
Many thanks in advance