Molecular Sizing using Fluorescence Correlation Spectroscopy
2010
Size is one of the basic characteristics of any object and especially of molecules. It is directly and independently of other properties related to fundamental physical phenomena, like diffusion, for example. The size of molecules can change when they interact with other molecules (e.g. when they bind ions), or when the temperature, pH or chemical composition of their surrounding environment varies. Therefore, molecular size can be a very sensitive detector for the state of a molecule. For this reason, molecular sizing finds broad applications in physics, chemistry and biology. However, in most cases these applications demand an accuracy of size determination in the order of Angstrom. My work was focused on sizing of molecules with high-precision at pico- to nanomolar concentrations using fluorescence correlation spectroscopy. An advanced modification of this technique called dual-focus FCS allows measuring the absolute value of the diffusion coefficient and therefore the absolute size of a molecule. The precision of this technique was evaluated and exemplified by the sizing of different molecules, including common globular proteins. In addition the observed quantitative relation between molecular weight and measured diffusion coefficient is discussed. I set up a new method derived from fluorescence correlation spectroscopy that is able to measure rotational diffusion constants of macromolecules. This method is suited to study the rotational diffusion of macromolecules having rotational diffusion times of dozen to hundred nanoseconds, a time range where fluorescence anisotropy cannot be applied. Using the determined rotational diffusion coefficient, values of the hydrodynamic radius for a set of common globular proteins were obtained.
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