Question

“Protein A” is phosphorylated at one amino acid by a kinase, “protein B”, and dephosphorylated by a phosphatase, “protein C”.

You have purified all three of these proteins and want to determine the structural changes that occur when protein A is phosphorylated or dephosphorylated. Explain in detail how you would go about designing and conducting this investigation.

Answer 1

Phosphorylation and dephosphorylation are important posttranslational modifications of native proteins. Phosphorylation and dephosphorylation occurs at tyrosine residues in a protein by the enzymes kinase and phosphatase respectively.
These tyrosine phosphorylation and dephosphorylation events occur immediately (i.e., within minutes) after receipt of the extracellular signal and lead to subsequent and more widespread changes in the phosphorylation and dephosphorylation of serine and threonine residues in proteins and enzymes in the cytoplasm and nucleus. With few exceptions the kinases and phosphatases show strict specificity for either tyrosine or serine and threonine side chains in proteins.

By using the fluorogenic probes, the phosphorylation and dephosphorylation events can be identified. The probe binds preferentially to phosphorylated peptides and does not with dephosphorylated peptide. By using the fluorogenic probe binding test, the phosphorylation can be tested out

Phosphorylation alters the structural conformation of a protein, causing it to become activated or deactivated. By examining the concerned catalyzed activity of the phosphorylated and / or dephosphorylated protein also the phosphorylation and / or dephosphorylation can be tested out.