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Functional Membranes

Functional Membranes. Armağan Koçer Biosensing with channels Ile de Berder, 25 August 2007,. Mechanosensitive channels. Nature. External trigger. X. X. Membrane stretch. Goal of the project. Structure. Dr. Maarten van Roosmalen. Mechanosensitive channel of large conductance, MscL.

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Functional Membranes

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  1. Functional Membranes Armağan Koçer Biosensing with channels Ile de Berder, 25 August 2007,

  2. Mechanosensitive channels

  3. Nature External trigger X X Membrane stretch Goal of the project

  4. Structure Dr. Maarten van Roosmalen

  5. Mechanosensitive channel of large conductance, MscL Closed Open

  6. O R S C H S 3 O R+ trigger R+ R+ R+ R+ Charging the pore General principle of activation CH2SH

  7. off X pA off msec on Single molecule measurements: patch clamp Current signal

  8. pH 6 H pH-induced channel activation pH 8

  9. 100 90 80 70 60 release / % trigger trigger 50 40 30 20 10 0 0 5 10 15 20 25 30 35 40 45 50 55 Time / min Ensemble Measurements: fluorescent dye efflux assay

  10. 7.75 80 60 7.35 Relative % release 40 20 0 6.1 7.4 7.8 pH Tuning of the pH interval for the channel activity MscL-G22C monomer Koçer et al., Angew. Chem. Int. Ed. Vol. 45, 3126 – 3130, 2006

  11. O N O O O 1 0.5 0 Absorbance Wavelength (nm) 250 350 450 550 Light-induced channel activation MscL-G22C monomer

  12. 200 ms 50 pA 10 s 50 pA 0 mmHg Light-induced channel activation

  13. Merocyanine state 366 nm >460 nm N Light-induced reversible channel activation: Spiropyran state

  14. 5 s 20 pA a b c Light-induced reversible channel activation 1min 20 pA O mmHg Vis UV UV a b c UV Vis UV A. Koçer, M. Walko, W. Meijberg, B. L. Feringa Science 309, 755 – 758, 2005 A. Koçer, M. Walko, B. L. Feringa Nature Protocols 2(6),1426 – 1437, 2007

  15. pH < pKa pH > pKa +H Light triggered - pH induced channel activation

  16. Conclusions and Perspectives • An external control over MscL activation: • pH induced activation • Light induced activation • Time controlled light / pH-induced activation • High throughput analyses of modified MscL in a fluorescent efflux experiment. • New tool for understanding the gating of MscL channel • New opportunities for micro-nano delivery systems • New opportunities for micro-nano sensory systems

  17. HHHHHH Prof. Dr. George T. Robillard Dr. Wim Meijberg Prof. Dr. Ben L. Feringa Prof. Dr. Bert Poolman Dr. Ir. Hans Kanger Prof. Dr. Ir. Vinod Subramaniam Drs Martin Walko Drs Alma Dudia Erna Bulten $ BiOMaDe Technology Foundation NanoNed Netherlands Organization for Scientific Research (NWO-CW) Materials Science Centre (MSC plus) University of Groningen KP6 MediTrans

  18. 2 years Post doc position available www.nanoned.nl Flagship: Single Molecule Chemistry and Physics Project: Transmembrane Response Systems Contact: Dr. Armagan Kocer kocer@biomade.nl

  19. 100 80 60 % release 40 20 0 0 100 200 300 400 500 time (sec) MscL in Membrane patches covering microwells:II. MscL reconstitution and GUV preparation Liposomes:DPhPC:POPG:cholesterol (70:25:5 wt%)

  20. 100 80 60 % release 40 20 0 0 100 200 300 400 500 time (sec) MscL insertion into planar bilayers Liposomes of Azolectin:ergosterol:PE (70:20:10 M) trigger trigger

  21. MscL: Mechano sensitive channel of large conductance E.coli MscL: a turgor operated emergency valve for solute release and osmotic adjustment in E. coli

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