Determination of the Partition Coefficient of Protein-Membrane Reversible Association By FCS and PCH

Abstract

Protein-membrane reversible association is critical to many cell and physiological functions. In most cases, protein association to the membrane is not stoichiometric and is best described by the partition coefficient rather than the dissociation constant. Fluorescence Correlation Spectroscopy (FCS) is a suitable methodology for the determination of the partition coefficient (Kp) of proteins into membranes (1, 2). Using FCS, Kp is typically determined from the diffusion of lipid vesicles loaded with fluorescently-tagged proteins. However, the uneven distribution of protein onto vesicles and the presence of unbound fluorescently-tagged protein due to dissociation events or the coexistence of a non-membrane competent protein conformer biases the retrieved quantities. In this work we investigate the effects of unbound protein in Kp quantification by FCS using antibody-membrane association as a model. In addition, we discuss the likely distribution of proteins onto vesicles in the case of reversible protein-membrane association, when the conventionally used Poisson distribution does not fully describe association/dissociation events. We compare the experimentally determined Kp against the results obtained upon simulating the diffusion of vesicles loaded with fluorescently-tagged antibodies following different statistical distributions. Finally, we discuss the conditions in which the Photon Counting Histogram (PCH) can be used to quantify the distribution of proteins associated to the vesicles. (1.) Melo, A. M., M. Prieto, and A. Coutinho. 2011. The effect of variable liposome brightness on quantifying lipid-protein interactions using fluorescence correlation spectroscopy. Biochimica Et Biophysica Acta-Biomembranes 1808(10):2559-2568. (2.) Kruger, D., J. Ebenhan, S. Werner, and K. Bacia. 2017. Measuring Protein Binding to Lipid Vesicles by Fluorescence Cross-Correlation Spectroscopy. Biophys. J. 113(6):1311-1320.