Oberflächen-Plasmon-Resonanz spektroskopische Studie an verschiedenen Biosensoroberflächen: Charakterisierung von Protein-Peptid und Protein-Lipid Wechselwirkungen
Oberflächen-Plasmon-Resonanz spektroskopische Studie an verschiedenen Biosensoroberflächen: Charakterisierung von Protein-Peptid und Protein-Lipid Wechselwirkungen
Surface plasmon resonance spectroscopy (SPR spectroscopy) is a technique for the time-resolved measurement of interactions between macromolecules. For thesemeasurements, it is necessary to immobilize a binding partner on the surface of asensor chip. The measurements are recorded as sensorgrams. In t...
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Personal Name(s): | Fischer, Torsten (Corresponding author) |
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Contributing Institute: |
Publikationen vor 2000; PRE-2000; Retrocat |
Imprint: |
Jülich
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
2000
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Physical Description: |
IX, 129 p. |
Document Type: |
Report Book |
Research Program: |
Addenda |
Series Title: |
Berichte des Forschungszentrums Jülich
3839 |
Link: |
OpenAccess OpenAccess |
Publikationsportal JuSER |
Surface plasmon resonance spectroscopy (SPR spectroscopy) is a technique for the time-resolved measurement of interactions between macromolecules. For thesemeasurements, it is necessary to immobilize a binding partner on the surface of asensor chip. The measurements are recorded as sensorgrams. In the present study,different surfaces were tested. The biological model system for the investigation ofinteractions was the binding of calmodulin to a synthetic peptide which correspondsto the amino-acid sequence of the CaM binding site from the typed nitrogen oxidesynthase. For binding CaM to this peptide, NOS-I WT, the rate constants (k$_{a}$, k$_{d}$) andthe equilibrium constants (KD) were determined by different evaluation methods.The dissociation constants determined from the sensorgrams for the different sensorchip surfaces increased with rising immobilization density. An analysis of the rateconstants determined from the sensorgrams showed that especially k$_{a}$ decreasedsubstantially with increasing immobilization density. The sensorgrams on the differentsensor surfaces could no longer be explained by a monoexponential bond model athigh immobilization density. This was also reflected in the decreasing stoichiometryof peptide NOS-I WT for CaM at increasing immobilization density of the peptide. Thebest agreement with the literature values achieved on a dextran surface at very lowimmobilization density (< 5.8 fmol/mm$^{2}$) on peptide NOS-I WT (K$_{D}$ values of 0.8 - 3.9nM).Furthermore, sensor chips with hydrophobic surfaces were produced to build upheterobilayers or phospholipid bilayers. These sensor chips were used to investigatethe Ca$^{2+}$-dependent membrane association of the Ca$^{2+}$ binding protein recoverin. AnN-terminal myristoyl group anchors recoverin at high calcium concentrations in themembrane. Mutants of recoverin were examined, whose affinities for Ca2+ ions weremodified in comparison to the wild type. All forms of recoverin (native, recombinantlymyristoylated (WT), EF+4, EF-3, EF-2) show a bond to the hydrophobic sensor chipsurface in the presence of Ca$^{2+}$ ions, although with different affinities (EC$_{50}$: nativerecoverin = 18 $\mu$M; WT = 5 $\mu$M; EF+4 = 29 $\mu$M; EF-3 = 8 $\mu$M; EF-2 = 9 $\mu$M).Interestingly enough, it was also possible to detect a bond to the phospholipid layerin the absence of Ca$^{2+}$ which, however, occurred with a markedly lower amplitude. |