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KINSC Supported International Research Program: Partnership with Lund University, Sweden

KINSC Supported International Research Program: Partnership with Lund University, Sweden Winnifred Johnson, 1 Catherine Divizio, 1 Mikael Bauer, 2 Sara Linse, 2 Karin Åkerfeldt 1 1 Department of Chemistry, Haverford College 2 Department of Biochemistry, Lund University, Sweden

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KINSC Supported International Research Program: Partnership with Lund University, Sweden

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  1. KINSC Supported International Research Program: Partnership with Lund University, Sweden Winnifred Johnson,1 Catherine Divizio,1Mikael Bauer,2 Sara Linse,2 Karin Åkerfeldt1 1Department of Chemistry, Haverford College 2Department of Biochemistry, Lund University, Sweden Calmodulin in Target Recognition

  2. Winn ‘09 Catherine ‘12 Summer of 2009: 5 weeks at Haverford + 5 weeks at Lund University

  3. Winnifred Johnson, Catherine Divizio, Mikael Bauer, Sara Linse, Karin Åkerfeldt Sara Mikael Catherine Erik Celia Winn

  4. Proteins How does structure relate to function?

  5. Calmodulin (CaM) • ubiquitous in eukaryotic cells • essential to cell function (excitability, motility, apoptosis, transcription, etc) • binds Ca2+ • mediates Ca2+ regulation • 1998: binds 40 peptide and protein targets • 2008: ca. 300 • 2009: ca. 300 + 72

  6. CaM Target binding CaM: blue Ca2+ : yellow ball target: red http://www.molmovdb.org/cgi-bin/morph.cgi?ID=180968-23252

  7. CaM 72 newly identified targets Sara Linse • protein array with proteins produced from a • human brain cDNA library (Ireland) • CaM tagged with a fluorescent chromophore • 10,000 unique proteins: 72 new targets • many membrane-associated and occur in • limited quantities

  8. CaM targets verify binding with synthetic peptides • Winn ‘09 started (senior research thesis), continued summer 2009 • identify potential CaM binding sites (typically 15-25 residues long) • synthesize, purify, verify identity and test Potassium voltage gated channel Kv6.1 (residues 474-493): QERVMFRRAQFLIKTKSQLS

  9. CaM targets verify binding with synthetic peptides green: hydrophobic blue: positively charged red: negatively charged Potassium voltage gated channel Kv6.1 (residues 474-493): QERVMFRRAQFLIKTKSQLS

  10. CaM Measure target binding: KV6.1(474-493) • Fluorescence spectroscopy (W, or other fluorescence chromophore) • Isothermal titration calorimetry (ITC), thermodynamic parameters • KD, DH, DG, DS • ITC measures the amount of heat generated or absorbed as one solution is titrated into another. If the solutions interact, there will be a change in heat. • A solution of peptide was titrated into CaM in the presence and absence of Ca2+.

  11. CaM CaM Measure target binding: KV6.1(474-493), ITC Peptide titrated into: CaM + Ca2+ CaM + EDTA buffer KD= 500 nM

  12. CaM Measure target binding: surface plasmon resonance, SPR Kinetic parameters, kon and koff http://www.fujifilm.com/products/life_science/other_product/ap3000/tech.html (100109)

  13. CaM Measure target binding: surface plasmon resonance, SPR linker-CQERVMFRRAQFLIKTKSQLS Potassium voltage gated channel Kv6.1 (residues 474-493): CQERVMFRRAQFLIKTKSQLS

  14. CaM What’s next? O’Connell DJO, Bauer MC, O’Brien J, Johnson WM, Divizio CA, O’Kane S, BerggårdT, Merino A, ÅkerfeldtKS, LinseS; Cahill DJ “Integrated protein array screening and high throughput validation of 70 novel neural calmodulin binding proteins” submitted for publication. Key question: How can calmodulin selectively regulate one protein in the presence of hundreds of others?

  15. CaM 1. Target recognition: parameters CaM: blue Ca2+ : yellow ball Differentiation can occur through CaM binding targets at different degrees of Ca2+ saturation Ca2+ binding affects both structure and charge of CaM

  16. CaM 2. Different modes in target binding CaM can bind targets in different ways and with different stoichiometries CaM: blue Ca2+ : yellow ball target: red

  17. Ca2+ 3.From fertilization to cell death • large Ca2+ concentration gradients (~10'000 to 1) between the extracellular fluid, cytoplasm, and internal calcium stores • Ca2+ used as cellular currency that supports the propagation of intracellular Ca2+ waves. • cells exploit the two key features of oscillatory signals - frequency and amplitude - to utilize Ca2+ as a second messenger to generate a large variety of intracellular signals. This is an efficient way to use the same second messenger to activate many different cell processes. Understanding both thermodynamic and kinetic binding parameters crucial kon koff KD Look at target binding under different Ca2+ and salt conditions (Mg2+) http://ki.se/ki/jsp/polopoly.jsp?d=18242&a=50201&f=sv&l=en (100109)

  18. Peptide Approach Key question: How can calmodulin selectively regulate one protein in the presence of hundreds of others? • Simplify. Proteins large, peptides small. Enable studies on targets that are difficult to come by • Peptide Approach enables us to verify new targets and learn more about their interactions with calmodulin • Build up a library of data (KD, kon and koff) to look for trends (look under different conditions, target binding under different Ca2+ concentrations, salt concentrations, Mg2+) • Use the peptides to do competition experiments • X-ray crystallography new CaM-peptide complexes in collaboration with Roger Rowlett, Professor of Chemistry, Colgate University • Ultimately, design experiments that will enable us to do this under Ca2+ oscillating conditions – pulse Ca2+ (cage Ca2+, release it upon photolysis)

  19. CaM Human versus plant sequences • CaM from eukaryotes completely conserved (100%)! • CaM in plants occur in multiple isoforms (Winn) Arabidopsis thaliana

  20. CaM Human versus plant sequences • Obtained clones of CaM from Arabidopsis thaliana from • Professor Ray Zielinski, Department of Plant Biology, University • of Illinois at Urbana-Champaign, Illinois • 3 isoforms purified to homogeneity (CaM4, CaM7, CaM9) • Ca2+ and target binding studies on-going Human and CaM9 different

  21. Summary • Project that addresses a key question of fundamental importance to • cell biology: Ca2+-regulated cell signaling mediated by calmodulin • Collaborative approach: chemical – using synthetic peptides a models • and a variety of biophysical instrumentation to determine target binding • The project is modular (synthesis, purification, analysis) and well suited for • undergraduate research at any level

  22. Island Ven (Hven)

  23. Tycho Brahe, Danish astronomer (1546-1601) 1576. Tycho Brahe received the island Hven from king Fredrik II Stjerneborg Tycho Brahe made a remarkable star catalogue of over 1000 stars. This was not the biggest catalogue in the number of stars, but in accuracy. His improvements of methods and accuracy in observations was very significant. He proved that comets are not objects in the atmosphere. He showed irregularities in the moons orbit. His wall quadrant and other instrument became widely copied and lead to improved stellar instruments. Kepler used Tycho Brahe's observations when he constructed his famous laws of planetary movement. http://www.nada.kth.se/~fred/tycho/index.html (100109) http://en.wikipedia.org/wiki/Wikipedia:Books/Tycho_Brahe/ (100109)

  24. Future?

  25. Acknowledgements Sara Linse and her group!

  26. Acknowledgements KINSC for monetary support!

  27. Acknowledgements YOU!

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