I have a question regarding which residues should I mutagenise into. By using bioinformatics method, I have identified some putative amino acid residues (Isoleucine, Methionine and Leucine) that may mediate this interaction.
However, the problem is that this interaction is potentially a hydrophobic interaction. Normally one would mutagenise something into Alanine to avoid side effect on the protein conformation. But Alanine itself is slightly hydrophobic, so I'm not sure if doing this can answer my question?
I can mutagenise it to something hydrophilic but I'm not entirely sure if this is a good idea because this can potentially change the protein conformation.
So what is your suggestion?
Any help would be appreciated.
Thank you
Dear Ann
there are examples of mutageneiys applied to hydrophobic surfaces of dimeric protein to induce monomerization.
For example in the past i worked with Human Cu,Zn Sod1 and for this proteins there is a characterized F50E/G51E/E133Q mutant Article The crystal structure of the monomeric human SOD mutant F50E...
) that is monomeric but active and structurally similar to the dimer.If there is the structure solved for your dimeric protein you can try to predict the effect that your hypothetical mutation will have on protein stability with bioinformatic tool. A simple (but in my experience powerfull tool) is POPmusic (Article PoPMuSiC 2.1: A web server for the estimation of protein sta...
) that is now available in DENZIME platform (https://soft.dezyme.com/). I used it in the past to design some stabilizing mutation with good result.However found the right mutant it is something not obvious and probably you will need to screen a panel of mutants and you will need to set up a protocol to check the protein stability/or activity. In this case i reccoment you PIPE CLONING approach for rapid generation of single point mutant with out the use of any specific mutagenesis kit.
A possible tool to check how much the mutation will affect the protein stability is in my opinion the DSF but of course only structural characterization or a positive result in a activity assay (if the monomer could be active ad the dimer) will prove you that mutation alter only the oligometizzation state and not the monomer folding,
Good luck
Manuele
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