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
Ca
Target Calmodulin
Ca
Ca
Ca
Bound Calmodulin
Target
Protein
Bound Calmodulin
Bound Protein
Bound Calmodulin
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 serinethreonine
phosphorylation/dephosphorylation
• Phosphorylase binds to calmodulin
Hypothesis
• Malencik and Anderson proposed
that calmodulin binding regions are
often sites of regulation by serinethreonine
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
Subtilisin
1
14
Hydroxylamine
CNBR
RXN
264
1
1
265
14
14
134
91
841
135
242
350
351
428
442
604
259
260
497
498
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
1 1- 91
11054.63
phosphorylation
2 92- 99
921.07
3 100-119 2184.45
4 120-147 2940.24
5 148-176 3229.75
6 177-224 5662.32
7 225-241 1938.17
8 242-350 12565.35
9 351-428 9322.85
10 429-441 1446.71
11 442-604 19129.32
12 605-615 1102.30
13 616-618 349.47
14 619-679 6480.42
15 680-682 425.57
16 683-692 991.20
17 693-699 735.79
18 700-713 1591.74
19 714-764 6134.86
20 765-766 263.38
21 767-800 4333.90
22 801-840 4545.12
pI
10.17 Residue that will be observed, serine-14 is site of
11.15
2.74
3.35
8.80
4.95
6.81
8.51
7.36
7.02
9.64
9.80
10.20
4.59
9.75
6.96
2.91
2.87
4.45
6.96
8.61
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
CNBR
RXN
1
14
91
242
350
351
428
442
604
Gel Filtration
Peptide
Fragments
Cation Exchange
1.0
CNBr separation of Peptides on SP-Sephadex
0.8
Absorbance 280 nm
Peptide 351-428
Peptide 1-91
0.6
0.4
Peptide 242-350 (unproven)
0.2
0.0
0
50
100
Tube Number
150
200
Amino Acid Analysis of
Glycogen Phosphorylase
Synthesized Peptide
1
91
14
MW= 11054.63
DQEKRKQISVRGLAGVENVT
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
-P
+P
Intact phosphorylase
+++
ND
CNBr digest (mixture)
+++
+
+
+
Peptide 1-91
++++
++
Synthetic peptide (6-25)
none
none
Glutathione (control)
none
none
Peptide 351-408
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