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@PHDTHESIS{vonderHocht:1302,
key = {1302},
othercontributors = {von der Hocht, Iris},
title = {{F}luorescence {S}pectroscopy of {R}ecoverin {F}unction and
{C}onformation},
volume = {4272},
issn = {0944-2952},
school = {Univ. Köln},
type = {Dr. (Univ.)},
address = {Jülich},
publisher = {Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag},
reportid = {PreJuSER-1302, Juel-4272},
series = {Berichte des Forschungszentrums Jülich},
pages = {119 p.},
year = {2008},
note = {Record converted from VDB: 12.11.2012; Köln, Univ., Diss.,
2008},
abstract = {Recoverin is a neuronal calcium sensor protein
(NCS-protein) from the vertebrate photoreceptor which is
involved in light adaptation. Recoverin changes its
conformation upon sequential binding of two calcium ions.
This conformational change induces the extrusion of a
covalently attached myristoyl residue from a hydrophobic
binding pocket which enables Recoverin to interact with
lipid membranes (calcium-myristoyl switch). In this thesis,
I report on my investigation of Recoverin’s calcium
myristoyl switch, using a recently developed modification of
fluorescence correlation spectroscopy (FCS) that is called
dual-focus FCS (2fFCS). This method allows for measuring
absolute diffusion coefficients with an accuracy of better
than a few percent. Recoverin, and the Recoverin mutants
E85Q and E121Q which bind only one or no Ca$^{2+}$,
respectively, were labeled with the fluorescent dye
Alexa647. Differences in the hydrodynamic radius due to
conformational changes of Recoverin and its mutants upon
calcium binding were monitored by measuring the diffusion
coefficient of these molecules as a function of free calcium
concentration. The calcium dependent interaction of
Recoverin with lipid membranes was measured in solutions of
small unilamellar vesicles (SUVs) of different lipid
composition. Again, diffusion measurements were used to
determine the fraction of free and lipid-bound Recoverin
using the strongly different diffusion coefficients of both
fractions. To account for the fluorescence brightness
difference of the dye label when in solution and close to a
lipid membrane, a new three-photon correlation analysis was
developed and tested.},
cin = {INB-1},
cid = {I:(DE-Juel1)VDB804},
pnm = {Grundlagen für zukünftige Informationstechnologien},
pid = {G:(DE-Juel1)FUEK412},
typ = {PUB:(DE-HGF)11 / PUB:(DE-HGF)3},
url = {https://juser.fz-juelich.de/record/1302},
}