Hi everyone,
Can i use Q61L Rac1 as a substitute for the GTP bound state of Rac1; as i came across a paper where they have mentioned that the Q61L Rac1 mimics the GTP bound state of Rac1.
Kindly share your experiences.
Thanks & Regards
Sunil Singh
It depends a lot upon your application. Q61L doesn't so much mimic the GTP bound state as it prevents GTPase activity. So if you've got mostly GTP in solution, it should bind GTP and never hydrolyze it. But if you've got mostly GDP, it should bind GDP instead - there's nothing preventing that.
Assuming you mean to use it in cells: it would be basically a 'constantly-on' Rac1 isoform. However, in our experience, 'always on' GTPases don't always give a very good readout for high GTPase activity in cells (like you'd get from overexpressing a GEF, for instance). Part of the problem seems to be that GTPases move around based on their bound nucleotide, and you lose that if you get rid of the ability to hydrolyze GTP. Fast cycling mutants, like Rac1 F28L, seem to give better/more realistic results in that regard.
But again, it depends upon your application. If you were looking for, say, proteins that specifically bound the GTP bound state, and planned to look via immunoprecipitation, Q61L would be perfect for that, since in the cellular environment it should rapidly become predominantly GTP bound, and it should stay that way until degraded.
Dear William,
Thank you so much for your answer. I have a protein which binds to GTP bound form of Rac1. I have to study their in-vitro binding affinity and for that i want GTP bound state of Rac1. Based on your answer, i assume i can use Q61L Rac1 mutant for my study.
Thank you so much
Regards,
Sunil Singh
Yes. You'll still need to add GTP to your buffers when you make the protein, to ensure that the nucleotide that goes in is GTP, but it should not hydrolyze, so it should maintain the GTP bound conformation more or less indefinitely.
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