Hi Manjeet, You are describing a rather complicated situation but you can try to sort it out in the following ways:
1. If you can make all the reagents i.e. your E3 ligase, all the putative E2s and E1, then you can do biochemical assays to figure out which E2's work. Boston Biochem sells E1 and a variety of E2's. The easiest assay would be a thio-ester release assay from E2. Basically you incubate E1 with E2, Ub and ATP to generate E2~S~Ub thioester intermediate which you can see on a non-reducing SDS-PAGE gel. Then you will have to block this reaction with NEM so that further charging of E2 is blocked. You can then test whether addition of your E3 and substrate results in the loss of Ub from the E2~S~Ub substrate. Please look into papers from Ray Deshaies lab to see how this is done. 2. Alternately you can test if any of the E2's make a stable complex with your E3. You can do this by a pull-down assay or you can incubate your E3 and the E2's and run them over a sizing column to see what makes a complex. 2. By Ub-E2-E3 complex do you mean a complex of E3 with a E2 which has a Ub linked to the E2 active site? If so, this has been done before by mutating the active site Cys of E2 to a Ser which substantially increases the stability of the E2~Ub ester bond. Regardless, you should always test this for your specific system. Try purifying the E2~Ub complex and then add the E3 and test if the complex is stable. It is perfectly possible that this will be a stable intermediate in the absence of substrate. I hope this helps. You can email me directly if you have further questions. Good luck! -Anirban On Tue, May 24, 2011 at 6:48 AM, manjeet mukherjee < [email protected]> wrote: > Hi all, > > I am working on a newly identified E3 ubiquitin ligase (RING type). I am > interested to co-crystallize it with E2 enzyme. Among the papers reported > so far, which are just a few, people have used a mixture of E2s including > UbcH1, UbcH5 and UbcH6 for the ubiquitination assays. However, there is no > clear evidence on E2 enzyme functions with this E3 ubiquitin ligase in vivo. > > > Can someone suggest how can I choose one particular E2 (among > UbcH1/UbcH5/UbcH6/UncH7...) for co-crystallization studies. It should be > noted that all these E2 enzymes have a similar fold, so will it make sense > to just randomly choose any one of these E2 enzymes and get started. > > Also, if someone with experience in this area suggest weather a Ub-E2-E3 > complex is stable enough and suitable for ternary complexes. > > Thanks in advance. > -Geet > -- Anirban Adhikari Postdoctoral Researcher Dept. of Biochemistry & Biophysics University of California San Francisco 600 16th Street Genentech Hall Room S414 San Francisco, CA 94158-2517 http://www.linkedin.com/in/anirbanadhikari
