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Src KinaseActivity upon
substrate phosphorylation
Outline 1.Src Kinase Introduction 2.Impacts of Src 3.Src reporter components
FPs (tECFP/EYFP) SH2 Flexible linker Substrate peptide4. Fluorescent Proteins and FRET5. Src Kinase Inactive and Active State6. How Src influence dynamical image of molecule in live cell7. Linker, Substrate designation for a robust labeling protein
Introduction of Src Kinase
• 1911 Peyton Rous isolated a virus from a chicken, which causes tumor in healthy bird, aka Rous sarcoma virus
• v-src codes for a protein which induces tumor cells.• c-src (cellular counterpart of v-src) affect signal
transduction pathway to regulate cell-growth• Despite external signals, v-src activates internal
control mechanism, hence induce oncogenic characterization.
Significant Impacts of Src activation
• Impacts on cell polarity, adhesion, focal adhesion assembly/disassembly, lamellipodia formation, and migration, survival of both normal cells and cancer cells.
• Inhibition of Src results in impaired polarization toward migratory stimuli
• Src phosphorylate cortactin. The phosphorylated cortactin associate and activate Arp2/3 to induce the growth of cortical actin network
Significant impacts of Src
• Src activates the calpain-calpastatin proteolytic system to cleave FAK and disrupt focal adhesion complex => cell adhesion to ECM is reduced and cell motility is enhanced.
• Src can phosphorylate p190RhoGAP and induce its binding to p120RasGAP => inhibition of RhoA, and subsequent dissolution of actin filaments.
• Because of Src’s prominent roles in invasion and tumor progression, epithelial-to-mesenchymal transition, angiogenesis, and the development of metastasis, Src is a promising target for cancer therapy.
Compositions of Src reporter
Fluorescent Proteins and FRET
• FPs: visualize signaling molecule– tECFP/EYFP pair
• FRET: visualize dynamical molecular activities.
How does FRET work?
• 2 chromophores are in proximity
• Overlap of excitation spectrum of donor and acceptor
• Energy transfer
Significance of flexible linker and substrate peptide
Src Kinase Structure
• Non-receptor tyrosine kinases family
• N-terminal SH4 domain• SH3 domain• SH2 domain (catalytic
domain)• C-terminal regulatory
sequence
How to activate Src Kinase?
1. Hormone binds cellular surface receptors (EGF, insulin) to generate phosphotyrosine
2. Phosphotyrosine attracts SH2 domain to activate src.
FRET effect of Src reporter upon the actions of Src Kinase and Phosphatase
Emission Spectra of Src reporter before(Red) and after(black) phosphorylation by Src
• When Src is inactivated, higher FRET is observed.
• When Src is activated, emission intensity drops, thus yields lower FRET efficiency
Various Src biosensors with tECFP at N-termini and Citrine at C-termini
Designation of a robust fluorescent labeling protein
Objectives• To compare the binding affinity(using MMPBSA/GBSA) between
phosphorylated complex (SH2 + phosphorylated peptide) vs. non-phosphorylated complex (SH2 + non-phosphorylated peptide)
• Create 5 prmtop files– Cplx1: SH2+linker+ phosphorylated peptide.– Cplx2: SH2+linker+peptide– Cplx3: linker+peptide– Cplx4: linker+ phosphorylated peptide– Cplx 5: SH2
• Run 20ns md-production • Plot Temperature, Energy, RMSD• Use MMPBSA to measure binding energy (delta G)
Cplx1 etot
Cplx2 etot
Cplx3 etot
Cplx4 etot
Cplx5 etot
Cplx1 temperature
Cplx2 temperature
Cplx3 temperature
Cplx4 temperature
Cplx5 temperature
Cplx1 rmsd
Cplx2 rmsd
Cplx3 rmsd
Cplx4 rmsd
Cplx5 rmsd
Cplx1(left) vs. Cplx2(right)
Cplx2 (left) vs. Cplx1(right)
GBSA of Cplx1• Differences (Complex - Receptor - Ligand):• Energy Component Average Std. Dev. Std. Err. of Mean• -------------------------------------------------------------------------------• BOND -0.2565 0.6121 0.0194• ANGLE -0.1049 0.4385 0.0139• DIHED 1.3343 0.2205 0.0070• VDWAALS -65.7397 5.9783 0.1891• EEL -1256.0463 39.3857 1.2455• 1-4 VDW 0.0000 0.0000 0.0000• 1-4 EEL 2.3653 1.2266 0.0388• EGB 1209.3920 34.8056 1.1007• ESURF -11.2648 0.5569 0.0176
• DELTA G gas -1318.4478 39.5743 1.2515• DELTA G solv 1198.1272 34.5976 1.0941
• DELTA G binding = -120.3205 +/- 9.1496 0.2893
GBSA of Cplx 2• Differences (Complex - Receptor - Ligand):
Energy Component Average Std. Dev. Std. Err. of Mean-------------------------------------------------------------------------------BOND -0.2331 0.6273 0.0198ANGLE -0.1340 0.4137 0.0131DIHED 1.4480 0.1877 0.0059VDWAALS -58.8694 4.9718 0.1572EEL -590.7593 40.2792 1.27371-4 VDW 0.0000 0.0001 0.00001-4 EEL 2.0730 1.2441 0.0393EGB 595.6172 35.9889 1.1381ESURF -9.1868 0.6921 0.0219
DELTA G gas -646.4749 39.7427 1.2568DELTA G solv 586.4304 35.7000 1.1289
DELTA G binding = -60.0445 +/- 7.9898 0.2527
PBSA of Cplx1• Differences (Complex - Receptor - Ligand):• Energy Component Average Std. Dev. Std. Err. of Mean• -------------------------------------------------------------------------------• BOND -0.2565 0.6121 0.0194• ANGLE -0.1049 0.4385 0.0139• DIHED 1.3343 0.2205 0.0070• VDWAALS -65.7397 5.9783 0.1891• EEL -1256.0463 39.3857 1.2455• 1-4 VDW 0.0000 0.0000 0.0000• 1-4 EEL 2.3653 1.2266 0.0388• EPB 1210.4326 35.0238 1.1075• ECAVITY -7.6109 0.2776 0.0088
• DELTA G gas -1318.4478 39.5743 1.2515• DELTA G solv 1202.8217 34.9004 1.1036
• DELTA G binding = -115.6261 +/- 12.3976 0.3920
PBSA of Cplx2• Differences (Complex - Receptor - Ligand):• Energy Component Average Std. Dev. Std. Err. of Mean• -------------------------------------------------------------------------------• BOND -0.2331 0.6273 0.0198• ANGLE -0.1340 0.4137 0.0131• DIHED 1.4480 0.1877 0.0059• VDWAALS -58.8694 4.9718 0.1572• EEL -590.7593 40.2792 1.2737• 1-4 VDW 0.0000 0.0001 0.0000• 1-4 EEL 2.0730 1.2441 0.0393• EPB 615.1288 37.0373 1.1712• ECAVITY -6.7999 0.4564 0.0144
• DELTA G gas -646.4749 39.7427 1.2568• DELTA G solv 608.3289 36.8231 1.1644
• DELTA G binding = -38.1460 +/- 7.9809 0.2524
Conclusion
• Substrate phosphorylation by Src Kinase would enhance binding affinity, and yield lower FRET response.