Dual-Focus Fluorescence Correlation Spectroscopy: Measuring Translational and Rotational Diffusion of Biomolecules

Abstract

We present numerous applications of the recently developed dual-focus fluorescence correlation spectroscopy (2fFCS) to the measurement of translational diffusion of proteins and protein complexes. The method is applied to measuring Ca2+-binding curves of wild-type and mutant of the proteins calmodulin and recoverin. 2fFCS is also capable of quantifying the conformational flexibility of macromolecular complexes, which is exemplified on the peptide binding of the major histocompatibility complex I (MHC I). Furthermore, we extended 2fFCS to measure fluorescence correlation at the nanosecond time scale, allowing also for measuring rotational diffusion. We performed a comparative study of translational and rotational diffusion (and related hydrodynamics size) of proteins, and present results for several globular proteins (BSA, human serum albumin, aldolase, ovalbumin). In all cases, measurements are performed at pico- to nanomolar sample concentrations, and with an accuracy of determining hydrodynamic size of a few percent. For performing 2fFCS measurements, a protein or protein complex needs only unspecific fluorescent labeling which is easily accomplished with commercially available dyes. Thus it is hoped that 2fFCS becomes a widely used and easy to handle technique in biophysical research.