Interaction of N-Interaction of N-terminal Peptides of terminal Peptides of

Glycogen Glycogen Phosphorylase with Phosphorylase with

CalmodulinCalmodulin

By James Proestos By James Proestos

Dr. Sonia Anderson’s LabDr. Sonia Anderson’s Lab

Biochemistry and Biophysics Biochemistry and Biophysics DepartmentDepartment

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

The Phosphorylated and The Phosphorylated and Unphosphorylated States of Unphosphorylated States of

Glycogen PhosphorylaseGlycogen Phosphorylase

Serine 14

Substrate(s)phosphorylase b phosphorylase a

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

Calmodulin Binding Calmodulin Binding ProcessProcess

4 Ca

ProteinProtein

2+

Cascade of Reactions in Cascade of Reactions in Glycogen DegradationGlycogen Degradation

Hormonal and Calcium control

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

Calmodulin/Phosphorylase B Calmodulin/Phosphorylase B InteractionInteraction

BoundCalmodulin/Sepharose

gel

Peptides that do not

bind to calmodulin

Rabbit Muscle Extract

SDS Page of Rabbit Muscle ExtractSDS Page of Rabbit Muscle Extract

96 K

68 K

42 K

29 K

18 K

12 K

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

HypothesisHypothesis• Malencik and Anderson proposed Malencik and Anderson proposed

that calmodulin binding regions that calmodulin binding regions are often sites of regulation by are often sites of regulation by serine-threonine 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 how the phosphorylation site and how does phosphorylation affect this does phosphorylation affect this binding to calmodulin?binding to calmodulin?

Phosphorylase PurificationPhosphorylase PurificationAmmonium Sulfate

Precipitation and Selective Crystallization

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

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

Cleavage of Cleavage of Phosphorylase BPhosphorylase B

1 84114

CNBR RXN

1

242

442

91

350

604

14

351 428

Peptide 1-91 PurificationPeptide 1-91 PurificationCation ExchangeCation Exchange

Synthetic Peptide 5-20Synthetic Peptide 5-205 14 20

SNQQLKRQISNQQLKRQISSVRGLAGVRGLAG

-P +P

Synthetic Peptide 5-20Synthetic Peptide 5-205 14 20

SNQQLKRQISNQQLKRQISSVRGLAGVRGLAG

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

Fluorescence TitrationFluorescence Titration

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 --

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 Phosphorylation of this peptide weakens the interaction with calmodulinthe interaction with calmodulin

• Peptide 1-91 binds more tightly to Peptide 1-91 binds more tightly to calmodulin than does peptide(5-20) (µM calmodulin than does peptide(5-20) (µM vs. nM)vs. 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

SRPLSRPLSDQEKRKQISVRGSDQEKRKQISVRGLAGVENVTELLAGVENVTELKKNFNRKKNFNRHLHFTLVKDRNVATPRDYYFHLHFTLVKDRNVATPRDYYFALAHTVRDHLVGRWIRTQQHYYEALAHTVRDHLVGRWIRTQQHYYEKDPKRIKDPKRIYYLSLEFYMYYLSLEFYM

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