Regensburg 2010 – wissenschaftliches Programm

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BP: Fachverband Biologische Physik

BP 34: Posters: New Technologies

BP 34.12: Poster

Donnerstag, 25. März 2010, 17:15–20:00, Poster B2

Measuring rotational diffusion of proteins by fluorescence correlation spectroscopy — •Anastasia Loman, Ingo Gregor, and Joerg Enderlein — Third Institute of Physics "Biophysics", Georg-August-Universität, Göttingen

Translational and rotational diffusion are thermally driven processes which depend on molecular parameters as size and shape but also on interaction between molecule and solvent environment. Fluorescence correlation spectroscopy (FCS) is a well known technique to measure translational diffusion coefficients of fluorescent molecules thus monitoring intramolecular changes and intermolecular interactions.

Here we apply fluorescence correlation to measure rotational diffusion. In contrast to conventional fluorescence anisotropy measurements, a correlation based method will work also when the rotational diffusion time is much longer than the fluorescence decay time. Thus, the method is ideally suited to study the rotational diffusion of macromolecules in aqueous solutions having rotational diffusion times of dozen to hundred nanoseconds. By using a pulsed interleaved excitation scheme with crossed excitation polarization, we are able to maximize the temporal dynamics of the measured correlation curve as caused by rotational diffusion. The method is exemplified on sizing the large globular proteins such as amylase, ovalbumin and human serum albumin.