Actually, Julian, to make Tyr non-phosphorylatable, a more conservative mutation would be to Phe instead of Ala since this would preserve most of the structure of the side chain. Ala would be a good choice for mutating a phosphorylated Ser/Thr.

A phosphomimetic mutation of Tyr is more difficult to achieve. Although you can attempt a mutation to Asp or Glu, the structure of either of these side chains differs significantly from that of phosphotyrosine, as does the charge density. Unfortunately, there is no better alternative; just be aware that the mimicking a phosphotyrosine is hard to achieve. Good luck Apurva.

Exchange the tyrosin residue to an alanin by site-directed mutagenisis and you'll get a Phospho-negativ mutant. A Phospho-mimic (active) you can receive after Exchange of alanin to aspartic acid or glutamic acid

Actually, Julian, to make Tyr non-phosphorylatable, a more conservative mutation would be to Phe instead of Ala since this would preserve most of the structure of the side chain. Ala would be a good choice for mutating a phosphorylated Ser/Thr.

A phosphomimetic mutation of Tyr is more difficult to achieve. Although you can attempt a mutation to Asp or Glu, the structure of either of these side chains differs significantly from that of phosphotyrosine, as does the charge density. Unfortunately, there is no better alternative; just be aware that the mimicking a phosphotyrosine is hard to achieve. Good luck Apurva.

Thanks all. Well I would like to define the real purpose of why I want to go for this constitutively active phospho tyrosine mutant protein.

Under certain experimental condition, the molecule on which I am working on undergoes dephosphorylation at a particular tyr residue. Hence, I want to know what happens when this dephosphorylation doesn't occur.

It's like when your molecule gets activated, you inhibit it and then see the comapratve analysis. For me it's reverse, it's getting inhibited and I want to see what's happening when it's inhbited versus when it's activated or atleast at basal level of activation.

I have one more option. I have an activator available in the market. I can use that. But since no activator/ inhibitor are completely foolproof as they might have some other unknown targets, so I am thinking for alternatives.

So, I was thinking if there can be some other way by which I can make this molecule free of inhibition such that it is atleast activated at the basal levels if not over activated. That's why I got through the idea of creating phosphotyrosine active mutant.

Please suggest me if I am thinking right and if there are other possible ways of solving my problem in a better way.

Apurva

Apurva, I don't believe there is a better way to do it than to mutate the Tyr of interest to both Glu and to Ala for comparison. Here is a relevant article on why this combination of mutations is about the best experiment you can do in vivo.

http://scienceblogs.com/scientificactivist/2009/11/12/on-mimicking-phosphotyrosine/

If I come across anything else, I will forward it to you.

Sure.. thanks .. I saw this blog already.. wasn't very sure abt this.. so thought to ask for some more suggestions here.. !!!

Apurva, do you know if there is a phosphatase that specifically dephosphorylates your phosphotyrosine residue of interest? Another way to go about this might be to try and block that dephosphorylation. To do so specifically, you might determine if the phosphatase is a sequence-specific, in which case you could mutate around the Tyr to block dephosphorylation.

I am happy to keep bouncing ideas back and forth if it helps you out. In my experience, generating phosphomimetic mutations usually involve trying all the combinations possible, and a good dose of luck.

If you can, let us know how it works out with the Tyr->Asp/Glu/Ala/Phe mutations!

Thanks Mark Chee... for all the help. I am going to discuss all such possibilities with my supervisor. Once I get through some thing, I will try to come back and discuss here... First I am going to try with the activator. If that doesn't work out the way I want it, then I will proceed to generating such mutants.

Good luck! Let's hope that activator is as specific as you need it to be.

You could try insertions if your fold is tolerant of this. Is the Tyr within a helix or beta strand or on a loop? If on a lopp why not try a flexible insertion, e.g. put in Gly-Glu-Phe-Glu-Gly – and check is this active?

You can actually direct insert good phospho Tyr mimic nowadays using in vivo unnatural amino acid incorporation.

Do tell Mingchao, how do you do this, and is it restricted to cell culture/bacteria/yeasts or can you do so in multicellular organisms as well?

I'm also interested in using an unnatural amino acid incorporation. How would that work? Auxotrophic strains?

Residue-specific incorporation of unnatural amino acids into proteins in vitro and in vivo

http://www.ncbi.nlm.nih.gov/pubmed/23423891

An unnatural amino acid that mimics phosphotyrosine

http://pubs.rsc.org/en/Content/ArticleLanding/2010/CC/c000283f#!divAbstract

Hello,

We have improved the method to incorporate the unnatural amino acid pCMF, a mimic of Tyr phosphorylation.

http://www.ncbi.nlm.nih.gov/pubmed/26329855

Regards

http://www.ncbi.nlm.nih.gov/pubmed/26329855