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Calmodulin and Phosphorylase Interaction

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... Interaction. By James Proestos. Biochemistry and Biophysics Department ... Department of Biochemistry and Biophysics. Kevin Ahern. Cleavage of Phosphorylase B ... – PowerPoint PPT presentation

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Title: Calmodulin and Phosphorylase Interaction


1
Calmodulin and Phosphorylase Interaction
  • By James Proestos
  • Biochemistry and Biophysics Department
  • Dr. Sonia Andersons Lab
  • Agriculture and Life Science Building

2
Calcium
  • Activator of many cellular processes (cell
    signaling)
  • Triggers muscle proteins to contract
  • Activates many enzymes

3
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 proteins
    activity.

4
Calmodulin Structure
5
Ca
Ca
Ca
Target Calmodulin
Ca
Ca
Ca
6
Bound Calmodulin
Ca
Ca
Ca
Ca
7
Bound Calmodulin
Ca
Target Protein
Ca
Ca
Ca
8
Bound Protein
Bound Calmodulin
Ca
Ca
Ca
Ca
9
Glycogen Phosphorylase Information
  • Found in fast twitch muscle tissue
  • It catalyzes the breakdown of glycogen
  • Controlled by phosphorylation/dephosphorylation

10
The Phosphorylated and Unphosphorylated States of
Glycogen Phosphorylase
B
A
Glucose
Serine
11
Cascade of Reactions in Glycogen Degradation
12
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

13
  • Phosphorylase binds to calmodulin
  • Hypothesis
  • Malencik and Anderson proposed that calmodulin
    binding regions are often sites of regulation by
    serine-threonine phosphorylation/dephosphorylation

14
  • 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?

15
Purification of Phosphorylase B
  • Grind rabbit muscle
  • Spin in a centrifuge
  • Remove the pellet
  • Ammonium sulfate precipitation and crystallize
  • Repeat crystallization several times

16
Phosphorylase Purification
  • Scan of phosphorylase gel

96 K
68 K
42 K
29 K
18 K
12 K
17
Phosphorylase Purification
18
Phosphorylase Purification
  • Scan of concentrated protein

19
Purification of Calmodulin
  • Grind bovine brain
  • Spin in centrifuge and remove pellet
  • Pass the supernatant through several columns

20
Purification of Calmodulin
21
Purification of Calmodulin
  • Scan of calmodulin gel

96 K
42 K
29 K
18 K
12 K
22
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).

23
Cleavage of Phosphorylase B
1
841
14
Hydroxylamine
Subtilisin
CNBR RXN
1
14
264
1
14
134
1
91
14
265
841
135
259
242
350
260
497
351
428
498
841
442
604
24
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

25
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
26
Separation of Glycogen Phosphorylase Peptides
  • Gel filtration
  • Separation based on size
  • Cation exchange
  • Separation based on charge (pI gt7)
  • High Performance Liquid Chromatography
  • Separation based on hydrophobicity
  • Analyze peptides
  • Peptide 1-91
  • Synthetic peptide 6-25
  • Calmodulin binding peptide

27
Cleavage of Phosphorylase B
1
841
14
CNBR RXN
1
91
14
242
350
351
428
442
604
28
Gel Filtration
Peptide Fragments
29
Cation Exchange
30
Amino Acid Analysis of Glycogen Phosphorylase
31
Synthesized Peptide
91
1
14
MW 11054.63
DQEKRKQISVRGLAGVENVT
MW 2228
32
HPLC Purification of Peptide 6-25
33
Mass Spectrometry of Peptide 6-25
34
Calmodulin/Phosphorylase B Interaction
Phosphorylase peptides
bound calmodulin gel
peptides that do not bind to calmodulin
35
Analysis of Calmodulin/Glycogen Phosphorylase
Interaction
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
36
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
37
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

38
Acknowledgements
  • Howard Hughes Medical Institute
  • Dr. Sonia Anderson
  • Dean Malencik
  • Department of Biochemistry and Biophysics
  • Kevin Ahern

39
Cleavage of Phosphorylase B
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