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Interaction of N-Interaction of N-terminal Peptides of terminal Peptides of
Glycogen Glycogen Phosphorylase with Phosphorylase with
CalmodulinCalmodulinBy James Proestos By James Proestos
Dr. Sonia Anderson’s LabDr. Sonia Anderson’s LabBiochemistry and Biophysics Biochemistry and Biophysics
DepartmentDepartment
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Glycogen Phosphorylase Glycogen Phosphorylase InformationInformation
• Found in fast Found in fast twitch muscle twitch muscle tissuetissue
• It catalyzes the It catalyzes the breakdown of breakdown of glycogenglycogen
• Controlled by Controlled by phosphorylation/ phosphorylation/ dephosphorylationdephosphorylation
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The Phosphorylated and The Phosphorylated and Unphosphorylated States of Unphosphorylated States of
Glycogen PhosphorylaseGlycogen Phosphorylase
Serine 14
Substrate(s)phosphorylase b phosphorylase a
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Calmodulin StructureCalmodulin Structure• Is found in all Is found in all
animal and plant animal and plant tissuestissues
• Binding of calcium Binding of calcium controls its ability controls its ability to bind to a to bind to a protein to regulate protein to regulate the target the target protein’s activityprotein’s activity
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Calmodulin Binding Calmodulin Binding ProcessProcess
4 Ca
ProteinProtein
2+
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Cascade of Reactions in Cascade of Reactions in Glycogen DegradationGlycogen Degradation
Hormonal and Calcium control
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The Interaction of Proteins The Interaction of Proteins in Glycogen Cascadein Glycogen Cascade
• Phosphorylase kinase becomes Phosphorylase kinase becomes active by calcium binding to the active by calcium binding to the intrinsic calmodulinintrinsic calmodulin
• The phosphorylase kinase The phosphorylase kinase interacts with the glycogen interacts with the glycogen phosphorylasephosphorylase
• It is not known if the calmodulin It is not known if the calmodulin can readily bind with glycogen can readily bind with glycogen phosphorylase in this interactionphosphorylase in this interaction
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Calmodulin/Phosphorylase B Calmodulin/Phosphorylase B InteractionInteraction
BoundCalmodulin/Sepharose
gel
Peptides that do not
bind to calmodulin
Rabbit Muscle Extract
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SDS Page of Rabbit Muscle ExtractSDS Page of Rabbit Muscle Extract
96 K
68 K
42 K
29 K
18 K
12 K
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HypothesisHypothesis• Malencik and Anderson Malencik and Anderson
proposed that calmodulin proposed that calmodulin binding regions are often sites binding regions are often sites of regulation by serine-of regulation by serine-threonine threonine phosphorylation/dephosphorylaphosphorylation/dephosphorylationtion
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HypothesisHypothesis• Malencik and Anderson Malencik and Anderson
proposed that calmodulin proposed that calmodulin binding regions are often sites of binding regions are often sites of regulation by serine-threonine regulation by serine-threonine phosphorylation/dephosphorylatiphosphorylation/dephosphorylationon
QuestionQuestion• Is the calmodulin binding region Is the calmodulin binding region
of phosphorylase b the same as of phosphorylase b the same as the phosphorylation site and the phosphorylation site and how does phosphorylation affect how does phosphorylation affect this binding to calmodulin?this binding to calmodulin?
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Phosphorylase PurificationPhosphorylase PurificationAmmonium Sulfate
Precipitation and Selective Crystallization
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Purification of CalmodulinPurification of Calmodulin• SDS Page of stages in calmodulin SDS Page of stages in calmodulin
purificationpurification• Four column chromatographies; 3000 fold Four column chromatographies; 3000 fold
purificationpurification
96 K
42 K 29 K 18 K
12 K
68 K
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Cleavage of Cleavage of Phosphorylase BPhosphorylase 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
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Cleavage of Cleavage of Phosphorylase BPhosphorylase B
1 84114
CNBR RXN
1
242
442
91
350
604
14
351 428
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Peptide 1-91 PurificationPeptide 1-91 PurificationCation ExchangeCation Exchange
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Synthetic Peptide 5-20Synthetic Peptide 5-205 14 20
SNQQLKRQISNQQLKRQISSVRGLAGVRGLAG
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-P +P
Synthetic Peptide 5-20Synthetic Peptide 5-205 14 20
SNQQLKRQISNQQLKRQISSVRGLAGVRGLAG
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Determine Affinity of
calmodulin-peptide complex by the use of dansyl calmodulin
fluorescence
Phosphorylate peptides and
recheck affinity
Isolated peptides
A)Peptide(1-91)B)Peptide(5-20)C)CaM Binding Peptide(s)
Analysis of Analysis of Calmodulin/Glycogen Calmodulin/Glycogen
Phosphorylase InteractionPhosphorylase Interaction
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Fluorescence TitrationFluorescence Titration
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Analysis of Peptide-Analysis of Peptide-Calmodulin InteractionsCalmodulin Interactions
Peptide Affinity -P +P
1-91 40 nM 60 nM
5-20 93 uM 225 uM
Selenoprotein W 18 nM --(KFRKLVTAIKAALAQ)
Melittin <1 nM --
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ConclusionConclusion• The N-terminal peptide(5-20) of The N-terminal peptide(5-20) of
phosphorylase binds to calmodulinphosphorylase binds to calmodulin• Phosphorylation of this peptide weakens the Phosphorylation of this peptide weakens the
interaction with calmodulininteraction with calmodulin
• Peptide 1-91 binds more tightly to Peptide 1-91 binds more tightly to calmodulin than does peptide(5-20) (µM vs. calmodulin than does peptide(5-20) (µM vs. nM)nM)
• The affinity of peptide 1-91 compared to 5-The affinity of peptide 1-91 compared to 5-20 suggests that additional sequences in 20 suggests that additional sequences in phosphorylase participate in calmodulin phosphorylase participate in calmodulin bindingbinding
SRPLSRPLSDQEKRKQISVRGSDQEKRKQISVRGLAGVENVTELLAGVENVTELKKNFNRKKNFNRHLHFTLVKDRNVATPRDYYFALHLHFTLVKDRNVATPRDYYFALAHTVRDHLVGRWIRTQQHYYEAHTVRDHLVGRWIRTQQHYYEKDPKRIKDPKRIYYLSLEFYMYYLSLEFYM
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AcknowledgementAcknowledgement
Howard Hughes Medical Institute Howard Hughes Medical Institute Dr. Sonia AndersonDr. Sonia Anderson Dean MalencikDean Malencik Andy BaumanAndy Bauman Department of Biochemistry and Department of Biochemistry and BiophysicsBiophysics Kevin AhernKevin Ahern