Calmodulin and Phosphorylase Interaction

By James Proestos Biochemistry and Biophysics

DepartmentDr. 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

Target Calmodulin

CaCa

Ca

Ca

Ca

Ca

Bound Calmodulin

Ca

CaCa

Ca

Bound Calmodulin

Ca

CaCa

Ca

Target Protein

Bound Calmodulin

Ca

CaCa

Ca

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

Serine

GlucoseA

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 84114

CNBR RXN

HydroxylamineSubtilisin

1 14 264

265 841

1 14 134

135 259

260 497

498 841

1

242

442

91

350

604

14

351 428

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 BNum 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 84114

CNBR RXN

1

242

442

91

350

604

14

351 428

Gel FiltrationPeptide Fragments

Cation Exchange

Tube Number

0 50 100 150 200

Absorbance 280 nm

0.0

0.2

0.4

0.6

0.8

1.0

CNBr separation of Peptides on SP-Sephadex

Peptide 351-428

Peptide 1-91

Peptide 242-350 (unproven)

Amino Acid Analysis of Glycogen Phosphorylase

Synthesized Peptide

1 9114

DQEKRKQISVRGLAGVENVT MW= 2228

MW= 11054.63

HPLC Purification of Peptide 6-25

Mass Spectrometry of Peptide 6-25

Calmodulin/Phosphorylase B Interaction

boundcalmodulin

gel

peptides that do not

bind to calmodulin

Phosphorylase peptides

Determine affinity of

calmodulin-peptide complex

Phosphorylate peptides and

recheck affinity

Isolated peptides

A)Peptide(1-91)B)Peptide(6-25)C)CaM Binding Peptide

Analysis of Calmodulin/Glycogen

Phosphorylase Interaction

Analysis of Phosphorylation Interaction

Peptide -P +P

Intact phosphorylase +++ ND

CNBr digest (mixture) +++ +

Peptide 351-408 + +

Peptide 1-91 ++++ ++

Synthetic peptide (6-25) none none

Glutathione (control) 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