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Proceedings Paper

Monitoring of small conformational changes by high-precision measurements of hydrodynamic radius with 2-focus fluorescence correlation spectroscopy (2fFCS)
Author(s): Thomas Dertinger; Iris von der Hocht; Ingo Gregor; Konstantin Komolov; Karl-Wilhelm Koch; Jörg Enderlein
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Paper Abstract

We report on our application of a new fluorescence-correlation spectroscopy technique, 2-focus FCS, for measuring the hydrodynamic radius of molecules with sub- Ångstrøm precision. The method is applied of monitoring conformational changes of proteins upon ion binding. In particular, we present measurements on Ca2+-binding of recoverin. Recoverin belongs to the superfamily of EF-hand Ca2+-binding proteins and operates as a Ca2+-sensor in vertebrate photoreceptor cells, where it regulates the activity of rhodopsin kinase GRK1 in a Ca2+-dependent manner. The protein undergoes conformational changes upon Ca2+-changes that are reflected as changes in their hydrodynamic radius. By using 2fFCS we were able to resolve hydrodynamic radius changes of ca. one Ångstrøm and used the Ca2+ dependence of this radius for recording binding curves in solution. We compare our results with those obtained by other techniques.

Paper Details

Date Published: 14 February 2007
PDF: 7 pages
Proc. SPIE 6444, Ultrasensitive and Single-Molecule Detection Technologies II, 64440H (14 February 2007); doi: 10.1117/12.704930
Show Author Affiliations
Thomas Dertinger, Forschungszentrum Jülich (Germany)
Iris von der Hocht, Forschungszentrum Jülich (Germany)
Ingo Gregor, Forschungszentrum Jülich (Germany)
Konstantin Komolov, Univ. of Oldenburg (Germany)
Moscow State Univ. (Russia)
Karl-Wilhelm Koch, Univ. of Oldenburg (Germany)
Jörg Enderlein, Forschungszentrum Jülich (Germany)

Published in SPIE Proceedings Vol. 6444:
Ultrasensitive and Single-Molecule Detection Technologies II
Jorg Enderlein; Zygmunt K. Gryczynski, Editor(s)

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