Julia Braunagel

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					   Max-Planck-Institut für Polymerforschung
   Max Planck Institute for Polymer Research




      Lysine-modified
valinomycin as a biosensor

                      Julia Braunagel



                          Ile de Berder
                           28.08.2007
Outline



1. Membrane model systems

2. Tethered bilayer lipid membranes

3. Cornell´s biosensor

4. Valinomycin as a biosensor
Model systems



Vesicles
                    sBLM‘s




 BLM‘s

                 tBLM‘s




                Picture: F. Giess
     Tethered bilayer lipid membranes, tBLMs




                                                                  Ion Channel


                                                                  Lipid Bi-Layer
Key features of a
suitable system:
                                                                  Tether
                                                                  (Spacerunit)
 -   Insulating
 -   Robust
                                                                  Substrate
 -   Stable
 -   Fluid
 -   Homogeneous

                              Cornell et al., Nature, 387 (1997) p. 580
                              Terrettaz et al., Angew. Chem. 40 (2001) p.1740
                              Schiller et al., Angew. Chem., 42(2003) p.208
                              Naumann et al, Langmuir, 19(2003) p. 5435
                              Atanasov et al., Biophy. J. 89 (2005) p. 1780
                              Köper et al, Advances in Planar Lipid Bilayers (2006)
Preparation of a tBLM
     A generic tBLM system
                                                  HO




                                             HO                    Phyt
                                                       O

                                                  O
                       Lipid

                                                                   DPG


                     Spacer                                O               n = 4, 6, 8, 12, 14
                                                               n


                     Anchor                                        X = Lipoic Acid, SH: Au, Ag,
                                                                          Cu, Fe, Ni, Pt, GaAs, Hg
                                                                   X = SiY: silicon oxide;
                                                       X                  (Y = Cl, OCH3, OC2H5)


V. Atanasov, et al.
Bioconjugated Chemistry 17, 631-637 (2006)
       DPTL, 2,3-di-O-phytanyl-sn-glycerol-1-tetraethylene glycol-D,L-α-lipoic acid ester
                                                                                      +           +
                                                                                N(CH 3)3+          N(CH 3)3+N(CH )           +
                                                                                                                              N(CH 3)3+N(CH )
                                                                                         N(CH 3)3               3 3+                       3 3
                                                                                                                    N(CH 3)3
                                                                              O                 O        O                 O
                                                                           O
                                                                             P O- O O       O
                                                                                               P O- O P -        O     O P - O O
                                                                                    P O-                    O O P -          O     P O-
                                                                            O                O                     O    O
                                                                                   O                  O                          O
                                                                             O                 O               O
                                                                                                                          O
                                                                         O O    O    O O O         O O O         O O O        O    O
                                                                               O O                O        O                 O O
                                          3.5 nm                                         O                 O O     O                  O


       Properties:

       -   R >10MW cm2
       -   C < 1µF cm-2
       -   Semi-Fluid
       -   Robust                                                                         O
                                                                O                                      O                         O            O
                                                                                                                    O
       -   Stable                         4.7 nm
                                                            O            O
                                                                             O        O
                                                                                          O
                                                                                                   O
                                                                                                       O
                                                                                                                O
                                                                                                                             O
                                                                                                                                 O
                                                                                                                                          O
                                                                                                                                              O
                                                                O            O                                      O
       -   Reproducible                                         O            O
                                                                                          O            O
                                                                                                                    O
                                                                                                                                 O            O

                                                            O                         O            O                         O            O
                                                                         O                                      O
                                                            O                         O            O                         O            O
                                                                         O                                      O
                                                        O                         O            O                         O            O
                                                                     O                O            O        O                O            O
                                                            O            O                                      O


                                                                             SS           SS                                     SS
                                         0.5 nm    SS           SS                                     SS               SS
                                                                                                                                                  Au
                                                                                                                                                  LaSFN9
Schiller et al., Angew. Chem., 42(2003) p.208
Patent : DE 102 22 588.5; EP 03 011 554.7; US-2004-0023304-A1
Available at diverchim.com
Incorporation of Gramicidin ion-channel


                              Cations e.g. K+




                              tBLM with Gramicidin




                              Gold
      Cornell´s biosensor


                                                 Streptavidin



                                                 Biotin




                                                 Tethered
                                                 membrane-
                                                 spanning
                                                 lipids




B.A. Cornell et al., 1997, Nature 387: 580-583
Valinomycin based biosensor
Valinomycin based biosensor



                              Advantages:
                              - easy synthesis on a
                                multi-milligram scale
                              - very specific to
                                potassium
                              - no tethered membrane-
                                spanning lipids
                                necessary
                              - no anchorage on the
                                surface necessary
Valinomycin based biosensor
     Valinomycin


                                             D-Hyiv
                             D-Val                                              Ester carbonyls are turned
                                         O
            L-Lac                                                               inwards, forming 6 ion-dipole
                                                     O
                             O           N
                                         H                            L-Val     bonds with a centrally situated
                O
                                 O               O                              cation.
                                                         HN       O
                    NH
    L-Val
                                                                        L-Lac
                         O
                O
                                     K+              O
                                                              O                 Modification on the backbone
D-Hyiv
                                                                                of the dodecadepsipeptide
                                                                      D-Val
            O       NH                               HN                         leads to the lost of complexing
                             O
                                             O                O                 properties.
                                     H
       D-Val             O           N           O                                    Exchange in the amino
                                                              D-Hyiv
                                 O                                                    acid sequence
                     L-Lac               L-Val




Shemyakin et al., 1974, FEBS Letters 44: 1-21
    Lysine-modified Valinomycin



                                              D-Hyiv
                              D-Val                                      NH2
                                          O
             L-Lac
                                                      O
                                                                                 Exchange of one L-Valin by
                                                                         L-Lys
                 O
                              O           N
                                          H                                      an L-Lysine
                                                  O
                                  O
                                                                   O
                     NH
                                                          HN                           possibility to bind e.g.
     L-Val
                                                                         L-Lac         biotin to valinomycin
                          O
                 O                    K+              O
                                                               O
 D-Hyiv
                                                                       D-Val
             O       NH                               HN
                              O                                O
                                              O
                                      H
      D-Val               O           N           O
                                                               D-Hyiv
                                  O
                      L-Lac               L-Val




The synthesis is described on my poster.
 Acknowledgment


Prof. W. Knoll

Dr. I. Köper

Dr. V. Atanasov




          Thank you for your attention!

				
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