Dresden 2009 – scientific programme

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BP: Fachverband Biologische Physik

BP 12: Single Molecules

BP 12.9: Talk

Wednesday, March 25, 2009, 12:15–12:30, HÜL 186

Dual-Focus Correlation Spectroscopy: Advantages and applications — •Anastasia Loman and Jörg Enderlein — III. Institut für Physik, Georg-August-Universität Göttingen, Friedrich-Hund-Platz 1, 37077 Göttingen

Fluorescence correlation spectroscopy (FCS) is a powerful technique for measuring diffusion coefficients of fluorescent molecules at pico- to nanomolar concentrations. A modified version of FCS, dual-focus FCS (2fFCS) shows significantly improvement in the reliability and accuracy of FCS measurements and allows for obtaining not relative but absolute values of diffusion coefficients [1].

The high precision of 2fFCS (absolute accuracy is shown to be better than 5 %) and the simple Stokes-Einstein relation directly coupled hydrodynamic radius and diffusion coefficient allow to monitor interactions of biomolecules - in particular proteins, RNA, DNA - with their environment or reacting to changes in environmental parameters such as pH, temperature, or chemical composition (e.g. protein unfolding) or performing biologically important functions (e.g. enzymatic catalysis).

We demonstrate that this method is sensitive enough to resolve length changes in small peptides of only one amino acis, and size changes of hydrodynamic radii as small as 0.5 nanometers. We used 2fFSC to study conformational changes of proteins such as phosphoglycerate kinase (PGK), α-amylase, and MHC class I complex under different conditions.

1. Dertinger, T. at al., ChemPhysChem 8 (2007) 433.