I need to find a mutant allele of my protein that is defective for homo-oligomerization, but can still bind a separate protein (heterodimer formation).
I think I will do alanine scanning mutagenesis of my protein.
However, my assays are very low throughput and I must assay each mutant protein individually. I simply cannot assay every single mutant allele in the time that I have.
I do not have any structural information. And Alphafold cannot solve the structure.
I am considering:
1. Mutate 3 residues at once. Is this too much? Will I simply destabilize my protein?
or
2. Only mutate charged residues, as oligomerization fequently relies on charged residues from my research. Does this seem incorrect?
Or is there better mutagenesis approach than alanine scanning?
Your suggestions seem adequate.
However, I would try mutating larger sections and then analyze the large sections by scanning alanine mutagenesis. For example, you can take a chunk of 10 or more amino acids and convert all to alanine. Walk this chunk throughout the entire protein sequence. Whichever chunk gives the phenotype you want, you should then study each section one amino acid at a time to see which amino acid(s) are involved with oligomerization.
Alternatively, you can delete section of the protein to find the oligomerization domain. I have seen papers where they fused different chunks of their polypeptide to GFP to identify which regions of the protein played a role into their phenotype.
It is not clear what assay do you have to test whether the homodimer interaction is impaired while retaining the heterodimer interaction. If you have an yeast-based assay (2 hybrid), you can mutagenize (use error-prone PCR conditions) your template, and then co-transform the linear PCR product and a wild-type plasmid (yeast powerful homologous recombination will recombine and you do not have to clone all your PCR products). After the first round of identifying mutants that disrupt the homodimer, you can test them against binding to the heterodimer. You can pick the colonies and identify the mutants by sequencing (attached reference with conditions).
Hope it helps,
a
Hi..
Alanine scanning mutagenesis is a useful approach for identifying the role of individual residues in a protein. It involves replacing each residue with alanine and observing the effect on protein function.
One possibility is to focus on mutating residues that are likely to be important for oligomerization. For example, you could look for residues that are conserved across related proteins that are known to oligomerize, or residues that are predicted to be involved in forming protein-protein interactions. You could also consider mutating residues that are predicted to be involved in stabilizing the protein, such as residues that are buried in the protein's core or residues that make important contacts with other residues in the protein.
It is generally not advisable to mutate more than one residue at a time, as this can significantly alter the protein's structure and make it difficult to interpret the results. However, if you have a specific reason to believe that mutating multiple residues at once will have a specific effect on protein function, it may be worth trying.
Another approach you could consider is yeast two-hybrid analysis. This method allows you to test whether a mutant protein can interact with another protein, and can be used to identify mutations that disrupt or enhance protein-protein interactions. However, it is worth noting that yeast two-hybrid analysis has some limitations and may not always accurately reflect the behavior of the protein in its native environment.
I hope this helps!
may be try first some deletions to see if the "heterodimer formation domain" and the homodimerization domain are separable. you can base your deletions on predicted structures/domain/motif of the protein ... may be it is not accurate but it is all you have !!! Any type of scanning needs a fast and easy test to be abble to screen. If you don't have such test then you need to proceed with a thoughtful step by step approach.
I think the homo-dimerization domain(s) (note it may not be one specific domain in the 2-dimensional protein but a patch in the folded protein) could be composed of charged residues, hydrogen bonding residues and perhaps even hydrophobic interactions. So be careful in presuming what it would look like.
The suggestion made by Mohammad Alzeyadi regarding yeast two-hybrid systems could be an interesting one. There are now many one and two hybrid interaction systems in yeast or in E. coli that you could look at. Using the wild type protein as both trap and bait should give you a positive signal through the homodimerization interaction, and then losing the signal could be your screen for mutants.
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