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Smart screen animations_Blue Sky Biotech
- 2. Families of membrane proteins •Single- and multi-pass transmembrane proteins
- 3. Families of membrane proteins •Single- and multi-pass transmembrane proteins •Peripheral membrane proteins Membrane Associated
- 4. Families of membrane proteins •Single- and multi-pass transmembrane proteins •Peripheral membrane proteins •Lipid-anchored (Farnesylated, prenylated, etc.) Membrane Associated Membrane Targeted
- 5. Families of membrane proteins •Single- and multi-pass transmembrane proteins •Peripheral membrane proteins •Lipid-anchored (Farnesylated, prenylated, etc.) Membrane Associated Membrane Targeted ?? Cytoplasmic
- 6. Assays for membrane proteins •Overexpressed recombinant catalytic fragments •Assay in solution; Highly disordered
- 7. Assays for membrane proteins • All cellular proteins present (complex) • Biologically relevant context • Laborious, slow, costly
- 8. Assays for membrane proteins Is there an assay system that: •Is simple to use? •Compatible with HTS? •Imparts the organization and context of a biological membrane? YES!
- 9. Assays for membrane proteins
- 10. Template Directed Assembly + His-tagged Proteins
- 11. Template Directed Assembly •HIS-tagged proteins adhere to fully fluid surface •Surface dictates biological organization: •topology and conformation •relationship to other subunits
- 12. Template Directed Assembly •HIS-tagged proteins adhere to fully fluid surface •Surface dictates biological organization: •topology and conformation •relationship to other subunits •Promotes self-assembly: •homo- and hetero- multimers Fluid surface Functional Assay
- 13. Template Directed Assembly •HIS-tagged proteins adhere to fully fluid surface •Surface dictates biological organization: •topology and conformation •relationship to other subunits •Promotes self-assembly: •homo- and hetero- multimers •higher-order complexes Fluid surface Functional Assay
- 14. Functional dimerization of Insulin RTK •Insulin receptor is normally activated through addition of high levels of manganese •HIS-tagged InR intracellular domain presented in context of TDA 2.0™ dimerizes and trans- phosphorylates in normal salt and Mn++ P- -P P- -P Template “First-Step”
- 15. Functional dimerization of Insulin RTK •Insulin receptor is normally activated through addition of high levels of manganese •HIS-tagged InR intracellular domain presented in context of TDA 2.0™ dimerizes and trans- phosphorylates in normal salt and Mn++ P- -P 7-fold increase in activity towards IRS1-derived peptide substrate! Template Template
- 16. TDA 2.0™ affect on other membrane kinases 200 100 50 25 10 5 1 [Enzyme] (nM)
- 17. Membrane context confers altered, relevant, substrate selectivity •TrkB was assayed against a random peptide library in the absence and presence of TDA 2.0™ • Substrate selection was remarkably different • Known substrates were only found in the presence of TDA 2.0™ Y1173 EGFR Y939 IRS1 Y987 IRS1 Y612 IRS1
- 18. Assembly of multi-component complexes •Significant literature suggesting that “accessory” proteins greatly affect enzyme pharmacology • TDA 2.0™ offers a simple way to assess enzyme activity in the proper context Insulin/IGF-1 Receptor EGF Receptor EGF/Her2
- 19. Assembly of multi-component complexes •Significant literature suggesting that “accessory” proteins greatly affect enzyme pharmacology • TDA 2.0™ offers a simple way to assess enzyme activity in the proper context Insulin/IGF-1 Receptor EGF Receptor EGF/Her2
- 20. Assembly of multi-component complexes •Significant literature suggesting that “accessory” proteins greatly affect enzyme pharmacology • TDA 2.0™ offers a simple way to assess enzyme activity in the proper context Insulin/IGF-1 Receptor EGF Receptor EGF/Her2
- 21. Assembly of multi-component complexes •Activation of Akt by mTOR and PDK1 on TDA 2.0™ pmol Phosphate Added 1 2 3 4 5 6 7 8 9 10 mTOR Anti-GST PDK1 Akt1 Anti-HIS Phospho-Akt1 Anti-Akt p-S473 TDA 2.0™ - + - - - + - + - + Akt1 + + - - + + + + + + Phospho-Akt1 Anti-Akt p-T308 PDK1 - - + - + + - - + + mTOR - - - + - - + + + +
- 22. Want TDA 2.0™ in your lab? TDA my way! Our SmartScreen™ kits contain pre- Our field applications scientific team (FAST) validated enzymes optimized with TDA 2.0™ can help implement TDA 2.0™ technology in your lab, or develop a customized assay to suit your application! www.blueskybiotech.com products@blueskybiotech.com