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14 February 2007 Monitoring of small conformational changes by high-precision measurements of hydrodynamic radius with 2-focus fluorescence correlation spectroscopy (2fFCS)
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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.
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Thomas Dertinger, Iris von der Hocht, Ingo Gregor, Konstantin Komolov, Karl-Wilhelm Koch, and Jörg Enderlein "Monitoring of small conformational changes by high-precision measurements of hydrodynamic radius with 2-focus fluorescence correlation spectroscopy (2fFCS)", Proc. SPIE 6444, Ultrasensitive and Single-Molecule Detection Technologies II, 64440H (14 February 2007); https://doi.org/10.1117/12.704930
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