Mutated a Arginine residue with Alanine by SDM in the active site and the protein became insoluble. WT protein was found soluble but the mutated one was found in pellet fraction.
Dear Eniyan,
It sems to me that you provide the answer to your own question : obviously, in your case, the single mutation you introduced in your protein is sufficient to make it insoluble. The behaviour of proteins is basically unpredictable; that's what makes them interesting
Dear Eniyan,
It sems to me that you provide the answer to your own question : obviously, in your case, the single mutation you introduced in your protein is sufficient to make it insoluble. The behaviour of proteins is basically unpredictable; that's what makes them interesting
A classic example of a change in the physical properties of a protein caused by a single amino acid change is sickle cell anemia, in which a single mutation in hemoglobin causes the protein to polymerize at low oxygen tension.
Did you check, if the mutated protein is folded? I suspect that the mutation causes your protein to misfold and hence become insoluble.
You could try other replacements that are not as radical, e.g. Lysine or Histidine. Maybe it'll keep your protein soluble (and perhaps even active)?
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