I made several point mutations on a protein, and found that only one particular Ser to Ala mutation leads to significant change in mobility of protein on SDS-PAGE (WB is attached). The drop in size is too large to suspect that is due to phosphorylation. Also when the same Ser is mutated to Asp there is slight change in mobility as well. Mutations of any other Ser did not change the mobility of protein.
Any explanations are welcome :)
This is an interesting result. Is this generated using an in vitro transcription/translation system or is this being produced in cells? Is this alteration in a signal sequence for example trafficking to golgi, so that post translational modifications at distal sites might be being impacted. This could also be true if it impacts membrane or docking protein interactions.
Frank R Burns thanks for your answer. the protein was over expressed in 293T (and two other mammalian cell lines- with the identical results). the Ser residue is in proximity to nuclear export sequence.
Is the particular serine residue glycosylated? Could explain shift in mobility when a mutation modulates glycosylation ability at a particular point on a protein.
Changes to an Asp can alter conformation of a protein by imitating phosphorylation and therefore an increase in size. The Serine residue itself looks as if this is modified in some way either phosphorylated or O link glycosylated. Removing these modifications by changing ser>Ala will change the conformation of the protein and therefore effect the analysis by SDS-PAGE compared to wild type which has retains a modified serine.
You can deglycosylate proteins using specific enzymes to test this and dephosphosphorylate proteins using alkaline phosphatase then analyse samples by SDS.
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