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.

Calmodulin Structure

Ca Ca Target Calmodulin Ca Ca Ca Ca

Bound Calmodulin

Bound Calmodulin Target Protein

Bound Calmodulin Bound Protein

Glycogen Phosphorylase Information • Found in fast twitch muscle tissue • It catalyzes the breakdown of glycogen • Controlled by phosphorylation/dephosphorylation

The Phosphorylated and Unphosphorylated States of Glycogen Phosphorylase B A Glucose Serine

Cascade of Reactions in Glycogen Degradation

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

Phosphorylase Purification

Phosphorylase Purification • Scan of concentrated protein

Purification of Calmodulin • Grind bovine brain • Spin in centrifuge and remove pellet • Pass the supernatant through several columns

Purification of Calmodulin

Purification of Calmodulin 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).

Cleavage of Phosphorylase B

1 14 841 1 14 265 Subtilisin 264 841 Hydroxylamine 1 14 CNBR RXN 91 242 351 442 350 428 604 1 14 135 134 259 260 498 497 841

Cleavage of Phosphorylase B Cyanogen bromide is our tool to cleave at methionine residues The resulting peptides will be the focus of the binding of calmodulin

Cleavage of Phosphorylase B Num From-To MW pI

1 1- 91 11054.63 10.17 Residue that will be observed, serine-14 is site of phosphorylation

2 92- 99 921.07 11.15 3 100-119 2184.45 2.74 4 120-147 2940.24 3.35 5 148-176 3229.75 8.80 6 177-224 5662.32 4.95 7 225-241 1938.17 6.81

8 242-350 12565.35 8.51

9 351-428 9322.85 7.36

10 429-441 1446.71 7.02

11 442-604 19129.32 9.64

12 605-615 1102.30 9.80 13 616-618 349.47 10.20 14 619-679 6480.42 4.59 15 680-682 425.57 9.75 16 683-692 991.20 6.96 17 693-699 735.79 2.91 18 700-713 1591.74 2.87 19 714-764 6134.86 4.45

20 765-766 263.38 6.96 21 767-800 4333.90 8.61

22 801-840 4545.12 10.20

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

1 14 841 1 14 CNBR RXN 91 242 351 442 350 428 604

Gel Filtration

Peptide Fragments

Cation Exchange

1.0

0.8

CNBr separation of Peptides on SP-Sephadex Peptide 351-428 Peptide 1-91

0.6

0.4

0.2

0.0

0 Peptide 242-350 (unproven) 50 100 Tube Number 150 200

Amino Acid Analysis of Glycogen Phosphorylase

1 14 Synthesized Peptide 91 MW= 11054.63

DQEKRKQI

S

VRGLAGVENVT MW= 2228

HPLC Purification of Peptide 6-25

Mass Spectrometry of Peptide 6-25

Calmodulin/Phosphorylase B Interaction Phosphorylase peptides bound calmodulin gel peptides that do not bind to calmodulin

Analysis of Calmodulin/Glycogen Phosphorylase Interaction Isolated peptides A)Peptide(1-91) B)Peptide(6-25) C)CaM Binding Peptide Determine affinity of calmodulin-peptide complex Phosphorylate peptides and recheck affinity

Analysis of Phosphorylation Interaction

Peptide Intact phosphorylase CNBr digest (mixture) Peptide 351-408 Peptide 1-91 Synthetic peptide (6-25) Glutathione (control) -P +++ +++ + ++++ none none +P ND + + ++ none none

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

Cleavage of Phosphorylase B