Experimental Dissection of Excluded Volume Effects from Quinary Interactions in Macromolecular Crowding

Abstract

Current models of diffusion are, at best, approximations of what happens in vivo. Fick's Law and Stokes-Einstein approximate dilute solution behavior, but they do not account for in vivo environmental factors that modulate particle motion such as electrostatic forces, hydrophobic interactions, and excluded volume. Such factors represent the contributions from macromolecular crowding that lead to diffusional anomalies seen in living cells. In the past, separation of excluded volume effects from the quinary interactions created by weak electrostatic and hydrophobic forces has been challenging. We are using a combination of fluorescence correlation spectroscopy (FCS) and microfluidic devices to tease apart these contributions such that they may be differentially quantified. FCS allows precise measurement of single molecule fluorescence thereby permitting assessment of local mobility in the presence and absence of crowders. By modulating the crowder surface area under conditions of constant excluded volume, quinary forces may be assessed and compared to the diffusional impact of excluded volume alone. Our long-term goal is to derive a more precise mathematical model for how a particle will diffuse in an intracellular environment. ∗EJB and VJA contributed equally to this work.