Interaction of N-terminal
Peptides of Glycogen
Phosphorylase with
Calmodulin
By James Proestos
Dr. Sonia Anderson’s Lab
Biochemistry and Biophysics
Department
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
phosphorylase b
phosphorylase a
Substrate(s)
Serine 14
Calmodulin Structure
•
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 Binding Process
2+
4 Ca
Protein
Cascade of Reactions in
Glycogen Degradation
Hormonal and Calcium control
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
Calmodulin/Phosphorylase B
Interaction
Rabbit Muscle
Extract
Bound
Calmodulin/
Sepharose
gel
Peptides
that do not
bind to
calmodulin
SDS Page of Rabbit Muscle Extract
96 K
68 K
42 K
29 K
18 K
12 K
Hypothesis
•
Malencik and Anderson proposed
that calmodulin binding regions are
often sites of regulation by serinethreonine
phosphorylation/dephosphorylation
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?
Phosphorylase Purification
Ammonium Sulfate Precipitation
and Selective Crystallization
Purification of Calmodulin
SDS Page of stages in calmodulin purification
• Four column chromatographies; 3000 fold
purification
•
96 K
68 K
42 K
29 K
18 K
12 K
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
1
14
841
CNBR
RXN
1
14
91
242
350
351
428
442
604
Peptide 1-91 Purification
Cation Exchange
Synthetic Peptide 5-20
5
14
SNQQLKRQISVRGLAG
20
Synthetic Peptide 5-20
5
14
20
SNQQLKRQISVRGLAG
-P
+P
Analysis of
Calmodulin/Glycogen
Phosphorylase Interaction
Determine
Affinity of
calmodulin-peptide
complex by the use
of dansyl calmodulin
fluorescence
Isolated peptides
A)Peptide(1-91)
B)Peptide(5-20)
C)CaM Binding Peptide(s)
Phosphorylate
peptides and
recheck affinity
Fluorescence Titration
Analysis of Peptide-Calmodulin
Interactions
Peptide
-P
Affinity
+P
1-91
40 nM
60 nM
5-20
93 uM
225 uM
Selenoprotein W
18 nM
--
(KFRKLVTAIKAALAQ)
Melittin
<1 nM
--
Conclusion
•
The N-terminal peptide(5-20) of phosphorylase
binds to calmodulin
•
Phosphorylation of this peptide weakens the
interaction with calmodulin
•
Peptide 1-91 binds more tightly to calmodulin
than does peptide(5-20) (µM vs. nM)
•
The affinity of peptide 1-91 compared to 5-20
suggests that additional sequences in
phosphorylase participate in calmodulin binding
SRPLSDQEKRKQISVRGLAGVENVTELKKNFNRHLHFTLVKDRNVATPRDYYFALAHT
VRDHLVGRWIRTQQHYYEKDPKRIYYLSLEFYM
Acknowledgement
Howard Hughes Medical Institute
Dr. Sonia Anderson
Dean Malencik
Andy Bauman
Department of Biochemistry and
Biophysics
Kevin Ahern