6. You are interested in a novel protein that you name Link. You know that Link
ID: 254784 • Letter: 6
Question
6. You are interested in a novel protein that you name Link. You know that Link (Molecular Weight: 100kDa) is located in the ER, but you would like to investigate its function as well as any post-translational modifications (PTMs) it may have. Your boss (who has a lot of experience studying PTMs), suggests introducing the following mutation into Link. The region of the Link with the mutation is shown below WT: Mutant Link: HVLRRPSFGGT HVLRRPAFGGT (highlighted is mutation). From a series of experiment, you determine that Link interacts with the protein Ganon (MW: 54kDa). Your boss suggests the following experiment which you dutifully perform: you incubate purified Link (WT or mutant) with Ganon in the presence or absence of PKA (a kinase) and/or ATP for an hour (note: proteins incubated in buffer with 20mM glycan). You run the sample on a gel and perform a Western blot with antibodies against Link and Ganon. The blot is shown below:Explanation / Answer
Wild Link contains serine involved in O-linked glycosylation in presence of PKA and ATP (as shown in the gel, molecular weight increases from 100 to 100.1kDa). Whereas mutated Link is not glycosylated due to mutation from serine to alanine (as shown in gel molecular weight not changed in presence of PKA and ATP). Due to the amino acid mutation mutant Link become inactive because of absence of glycosylation. Wild type Link (WT Link) has favoured the glycosylation of Ganon (molecular weight increased from 54 to 60 kDa) where in presence of mutated Link; there is no molecular weight increase in Ganon. (as shown in gel picture). Ganon have Asn (asparagine) amino acid involved in N-linked glycosylation.
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