Presentation on theme: "Calmodulin and Phosphorylase Interaction By James Proestos Biochemistry and Biophysics Department Dr. Sonia Anderson’s Lab Agriculture and Life Science."— Presentation transcript:
Calmodulin and Phosphorylase Interaction By James Proestos Biochemistry and Biophysics Department Dr. Sonia Anderson’s Lab Agriculture and Life Science Building
Calcium Activator of many cellular processes (cell signaling) –Triggers muscle proteins to contract –Activates many enzymes
Calmodulin Information Is found in all animal and plant tissues Binding of calcium controls its ability to bind to a protein to regulate the target protein’s activity.
The Interaction of Proteins in Glycogen Cascade Phosphorylase Kinase becomes active by calcium binding to the intrinsic calmodulin The phosphorylase kinase interacts with the glycogen phosphorylase It is not known if the calmodulin can readily bind with glycogen phosphorylase in this interaction
Phosphorylase binds to calmodulin Hypothesis Malencik and Anderson proposed that calmodulin binding regions are often sites of regulation by serine- threonine phosphorylation/dephosphorylation
Phosphorylase binds to calmodulin Hypothesis Malencik and Anderson proposed that calmodulin binding regions are often sites of regulation by serine- threonine phosphorylation/dephosphorylation Question Is the calmodulin binding region of phosphorylase b the same as the phosphorylation site and how does phosphorylation affect this binding to calmodulin?
Purification of Phosphorylase B Grind rabbit muscle Spin in a centrifuge Remove the pellet Ammonium sulfate precipitation and crystallize Repeat crystallization several times
Phosphorylase Purification Scan of phosphorylase gel 96 K 68 K 42 K 29 K 18 K 12 K
Scan of calmodulin gel 96 K 42 K 29 K 18 K 12 K
Experimental Plan Cleave the glycogen phosphorylase protein into peptides Isolate peptides of interest by conventional column chromatography Determine the binding of the peptides using a calmodulin affinity column Identify the peptide(s) from the calmodulin affinity column by mass spectrometry and/or amino acid analysis Phosphorylate the peptide(s) and compare its affinity for calmodulin to that of the unphosphorylated peptide (by fluorescence).
Separation of Glycogen Phosphorylase Peptides Gel filtration –Separation based on size Cation exchange –Separation based on charge (pI >7) High Performance Liquid Chromatography –Separation based on hydrophobicity Analyze peptides –Peptide 1-91 –Synthetic peptide 6-25 –Calmodulin binding peptide
Cleavage of Phosphorylase B 184114 CNBR RXN 1 242 442 91 350 604 14 351428
Future Work Determine and verify phosphorylation and stoichiometry of the various peptides by the use of ATP32 Determine the affinity of the peptides quantitatively by the use of fluorescence titration Complete sequence and/or mass spectroscopy information on the petide/s that bind to the solid support calmodulin column and, if enough material is isolated, to perform calmodulin binding Redetermine the binding of peptide 6-25 utilizing different approaches, if necessary, to verify that it does not bind to calmodulin Subfragment peptide 1-91 and redetermine its binding to calmodulin
Acknowledgements Howard Hughes Medical Institute Dr. Sonia Anderson Dean Malencik Department of Biochemistry and Biophysics Kevin Ahern