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Designing a protein that can autocatalytically cleave itself

  1. Oct 13, 2011 #1
    I've been given an 'assignment' of sorts (more of a thought exercise really) to mutate specific amino acid residues in a viral protein (from poliovirus) such that it can catalyse its own cleavage independently of RNA.

    I've learnt from my own research that the usual cleavage mechanism involves phosphate groups on the RNA backbone coordinating a zinc ion, and positioning it such that it coordinates oxygens in the carbonyl group of the scissile peptide bond, thereby polarising it. Once polarised, I assume the scissile bond is 'activated' for the subsequent steps of cleavage.

    I'm really confused as to how to replicate this mechanism in the absence of RNA and would appreciate any pointers (not answers necessarily, just hints, as I am totally lost!) The only vague guidance I've been given is something to do with the torsion angles of the amino acid backbone. Thanks in advance.... :)
     
  2. jcsd
  3. Oct 16, 2011 #2

    epenguin

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    You are asked to design a protease so think about proteases instead of remaining fixated on ribonucleases!

    Most proteases do digest themselves I think, at least the common digestive enzymes, though this is I think between two protein molecules, I do not know or remember about any of a molecule attacking a site in the same molecule which maybe is being asked but as I see no fundamental reason why not that probably only means I am not informed or up to date.
     
    Last edited: Oct 17, 2011
  4. Oct 17, 2011 #3
    Hi epenguin, thanks for the reply.

    I was referring to a protease - the RNA is only there to activate the protease. I'm just wondering whether the mechanism offered by RNA could be replaced by an internal mechanism for activation within the protein that is not dependent on RNA being there.

    As for self-cleaving proteins I know that inteins exist, I'm just not completely clued up on the exact mechanism...
     
  5. Oct 17, 2011 #4

    epenguin

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    Proteases generally are not dependent on RNA. Proteases cleave proteins. But then proteases are proteins.

    It seems you are being asked to look at an aminoacid sequence you are given and modify it into a protease. I suppose your given sequence has large homology with known proteases and you need to put a suitable residue/s at the active site or something like that?

    At first sight the question seems odd - if you make a protease that cleaves itself then you very soon won't have it any more! :confused:

    However you seem not to know or have forgotten about activation of the main digestive proteases by proteolysis of precursors wh is in all the biochemistry textbooks, then there are many others involved in stuff like apoptosis and cancer (metastasis) and other biol processes I am not familiar with. Here is someethingon a notorious viral protease http://en.wikipedia.org/wiki/HIV-1_protease . You seem a bit fixated on RNA through having worked on it but proteins are the smart molecules!
     
    Last edited: Oct 17, 2011
  6. Oct 17, 2011 #5

    Ygggdrasil

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    It seems like the OP is being asked to modify the poliovirus protease. Poliovirus expresses its proteins in a large polyprotein that gets cleaved by the poliovirus proteases (likely through intramolecular cleavage). These cleavages release the individual subunits from the polyprotein so that they can correctly assemble to form virions. Although I was not aware that the PV proteases depend on RNA, this could make sense as one of the proteases (poliovirus protein 3CD) is covalently attached to poliovirus's RNA-dependent RNA polymerase.

    This problem seems very hard and a lot of current cutting-edge research is aimed at trying to rationally re-engineer the activities and specificity of enzymes. Could you post a link to a paper so that I can read about the mechanism of this protease (both for my own curiosity about the enzyme and so maybe I can provide some help)?
     
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