So, I am working on a small protein (X) of Haloferax volcanii. It shows these results:
1. Mutant strain grows poorlycompared to wild type strain under low iron condition.
2. Growth of Mutant strain bettercompared to wild type under high iron condition.
With each subsequent passaging in low iron, growth of mutant strain keeps getting worse and worse.
3. Mutant strain showing less resistance to H2O2 stress.
4. It is surrounded by two iron related genes (dpsA, iron repressor).
My approach :
1. Looking at its translation level (westernblot) in low,standard and high iron.
2. Via northern blot, look for expression of neighbouring genes under mentioned iron conditions.
What else can I do??
some assays??
Will be grateful for your precious suggestions.
Thanks
Use BLAST to compare the sequence of the protein to other proteins. That may provide a clue to its function.
I think it is probable that the recombinantly expressed protein binds iron. You should probably find a colorimetric assay to observe and quantify bound iron. It is possible that coincubation with iron results in a wavelength shift. I think with a spectrophotometer you should collect the UV-vis spectra of
* the recombinant protein under a wide range of wavelength,
* a UV-vis spectra of just iron in the same buffer,
* a UV-vis spectra of the protein + iron
If you see a unique peak at a specific wavelength appear in the protein + iron condition, then with either constant concentration of iron and variable concentration of protein or the other case, if you measure the wavelength of the iron complex at variable concentrations then you should get a a binding curve of absorbance at the certain wavelength over a range of concentrations of either protein or iron. Then you can mathematically extract the Dissociation constant (KD) .
As for the purpose of iron, perhaps it is binding oxygen as is the case with hemoglobin which binds heme which contains a bound iron in the center that binds oxygen. The hemoglobin then carries the oxygen in the blood to all the essential organs. Perhaps the protein binds iron via an iron-sulfur cluster in which case maybe it facilitates electron transfer as the complexes in the mitochondrial electron transport chain do. Perhaps iron is an essential co-factor that catalyzes a chemical reaction of converting a certain substrate into a product.
Try AlphaFolding the sequence of your protein using collab notebooks:
https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb
Then use the PDB file generated from the structure prediction as structural search for all proteins of similar structure in the protein data bank using the DALI server: http://ekhidna2.biocenter.helsinki.fi/dali/index.html#tabs-1
You can also do a structural homology search with PDBeFold:
https://www.ebi.ac.uk/msd-srv/ssm/
If the structure prediction overlaps structurally sufficiently with a domain of a protein fished out of the protein data bank that shows iron bound in a crystal or cryo-EM structure then probably you would expect your protein has similar properties.
Also, would be kind of interesting if the wildtype and mutant strains respond differently to a magnet under low and high iron growth conditions. If that's the case then maybe the purpose of the protein is to be a magnetoreceptor for the organism to find iron deposits.
Is that a deletion or an expression mutant?
I would also do some alphaFold predictions. They can give you nice hints on the function. Metal binding proteins often do have very distinct structural domains.
AlphaFold multimer works also nice to predict potential protein interactions.
Small proteins often have regulatory funktion. Maybe its either transcriptional or posttranslational regulation of dpsA/DpsA. You could purify the small protein from H volcanii and check if you Co purify other proteins which match the neighbouring ORFs to check for protein protein interaction. And you could do qrtPCR to check for transription of dpsA and compare WT and mutant strain.
Tim Habenicht That is deletion mutant. but I do have expression mutant too, for analysis at translational level (Westernblot etc).
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