We often have good luck using protease cleavage sites as linkers. They're evolved to have certain flexibility to them and they usually have a healthy mixture of hydrophobic and hydrophilic amino acids. As a delightful bonus they also offer the option to separate the two partners at will.
Artem > I recently had some success in this area. My approach was to get lucky > when I guessed the linker. In this case, my linker became ordered > which seemed to help my fusion construct settle down enough to exhibit > density. My linker sequence came was (approximately) alternating > hydrophilic and hydrophobic residues. It ordered as part of a sheet > that mediated inter-protein contacts. I purposely did not use a low- > complexity motif for reasons I'll explain below. > > While staring at this structure and allowing my imagination to wander, > I came up with the idea of an "anything goes" linker motif. Find one > that can form an alpha helix or an extended conformation with equal > (in a subjective sense) regularity. The idea is that you want a > segment that can "adapt" to arbitrary crystal packing environments. I > think many natural segments that display multiple conformations have > been observed. Check out 1owr for a natural motif that can be alpha > helical or extended/disordered. (1owr is not the chimeric construct I > refer to above which is unpublished.) I think the natural linker in > 1owr would make an ideal linker in a chimeric construct. > > Although I base this advice on nothing but a hunch, stay away from > poly-gly or other absurdly simple motifs. The idea is purely > mathematical: think of choosing a linker as a guessing game, and you > want to guess the right linker. Let's pretend you want a 7 aa linker. > What are the chances that the optimal linker is going to be any random > sequence of residues? 20**7. So poly-gly, from a purely combinatorial > perspective, has a 1 in 20**7 chance to be the optimal sequence. But > the chances are probably lower than 1/20**7, because we know that poly- > gly is going to be disordered and the optimal sequence will probably > be ordered. In other words, common protein structure sense and > mathematics suggest that you will be shooting yourself in the foot if > you choose a poly-gly motif as a linker. > > An exception to the low complexity "rule" is the poly-A motif used in > fusion constructs with MBP. The poly-A is usually ordered and alpha > helical in these constructs. > > James > > On Apr 2, 2009, at 1:11 PM, Raji Edayathumangalam wrote: > >> Hi People, >> >> Could anyone point me to successful examples for two unrelated >> proteins that have been stitched together into one single >> polypeptide chain with flexible amino acids to create a functional >> chimera that was subsequently crystallized. I've looked up a few. >> >> I am particularly interested in understanding all the important >> considerations while designing a flexible linker even though many of >> these factors might be case dependent might be variable, obvious and >> commonsensical. Either way, I'd like to hear what folks have to say. >> >> Thanks very much. >> Raji >
