Zachary Ball et al show a new method for site-specific protein modifications using a dirhodium metallopeptide catalyst. This is a method that does not require cloning of the protein or organism of interest in order to introduce site-specific synthetic amino acids with linker groups as the side chain. In this case, tyrosine is the target for attachment of a biotin-diazo group. Only one tryptophan per protein is likely to be modified because the modified 'catalytic' protein will undergo protein-protein interactions which will spatially limit the amino acids in contact with the catalyst, thus allowing a local side-chain modification. This can be performed in biologically relevant buffers, tag a specific protein in E.coli lysate, and allow purification with the biotin group.
It seems like you would have to know a lot about your protein of interest for this to be useful. First you would have to know that it binds to a peptide. In this paper they had to synthesize their peptide catalyst, so the interacting peptide can not be very big. Also, you would have to know a lot about how this peptide binds, and whether a tryptophan residue is even present.
ReplyDeleteFigure 1 shows that the functional groups that can be modified are Trp, tyr, phe, asn, gln, asp, glu, arg, cys, his and ser. So, maybe I should have pointed this out, but they seem to focus on tryptophan in their actual experiment.
ReplyDeleteThey also say they have unpublished data that suggest it also works with other protein-peptide interactions.
The protein doesn't bind a peptide. The peptide is a modification to the protein of interest and then it is bound and modified by catalyst.
ReplyDeleteWhat kind of situation would this used?
You can't just throw in your catalyst without it being attached to a peptide - it would react with everything. The idea is that if you have a specific peptide-protein interaction, then you will have specific catalytic action.
ReplyDelete"For orienting our studies we examined heterodimerization of designed coiled coils, studied previously by our group, as a model of protein-peptide interaction for template modification." So, they're saying that this particular peptide-peptide interaction is just an example. The reason why they made the recombinant maltose binding protein with that E3gW peptide was because they had already modified the sequence and knew it interacted with K3RH2 in-vitro but wanted to test if they could use this method like a pull-down with thousand of other proteins in the mix.
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Here's how I see the steps necessary for application.
To get your metallopeptide, synthesize your peptide of interest and attach your dirhodium catalyst.(some sequence modification may be necessary for this attachment) The peptide then binds to its target protein. This brings the catalyst into proximity with the target. This will take a biotin-diazo group that has been added to the lysate, and stick it on your target. Then you can pull out your target, cut off the biotin, and identify the protein with mass spec. So, if you are starting off with a known or putative peptide ligand with an unknown receptor, you could use this method to find it.