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Two-Focus Fluorescence Correlation Spectroscopy: A versatile tool for precise measurements of molecular diffusion

Prof. Jörg Enderlein
Eberhard Karls Universität Tübingen


Thermally induced translational diffusion is one of the fundamental properties exhibited by molecules within a solution. Via the Stokes-Einstein relation it is directly coupled with the hydrodynamic radius of the molecules. Any change in that radius will change the associated diffusion coefficient of the molecules. Such changes occur to most biomolecules - in particular proteins, RNA and DNA - when interacting with their environment (e.g. binding of ions or other biomolecules) or performing biologically important functions (e.g. enzymatic catalysis) or reacting to changes in environmental parameters such as pH, temperature, or chemical composition (e.g. protein unfolding). Therefore, the ability to precisely measure diffusion coefficients has a large range of potential applications, for monitoring e.g. conformational changes in proteins upon ion binding or unfolding. However, many biologically relevant conformational changes are connected with rather small changes in hydrodynamic radius on the order of Ångstrøms. To monitor these small changes, it is necessary to measure the diffusion coefficient with an accuracy of better than a few percent. An elegant technique capable of measuring diffusion coefficients of fluorescent molecules at nanomolar concentrations is Fluorescence Correlation Spectroscopy (FCS) which was originally introduced by Elson, Magde and Webb in the early seventies. In its original form it was invented for measuring diffusion, concentration, and chemical/biochemical interactions/reactions of fluorescent or fluorescently labelled molecules at nanomolar concentrations in solution. However, standard FCS is prone to a wide array of optical and photophysical artefacts which make precise quantitative and absolute measurements of e.g. diffusion coefficients rather difficult. The main problem of standard FCS is the absence of a reliable extrinsic length scale in the measurements, which is, however, necessary for obtaining absolute values of the diffusion coefficient. Here, we report on our recently developed new technique of 2-focus fluorescence-correlation spectroscopy, allowing for measuring the hydrodynamic radius of molecules at pico- and nanomolar concentrations with sub-Angstrom precision. In 2fFCS, the problem of an extrinsic length scale is solved by generating two excitation foci with well defined distance from each other. Several applications of 2fFCS are presented, for example monitoring conformational changes of proteins upon ion binding, or monitoring protein unfolding curves upon chemical and thermal denaturation.