Macromolecular Crowding is a project of direct biological relevance. Macromolecules occupy 30% of the volume of the cell, strongly influencing inter-molecular interactions. Even in the absence of any direct interactions between particles, such as electrostatic, hydrophobic, or van der Waals forces, the macromolecules feel each others presence simply because two molecules cannot occupy the same place at the same time. This crowding of molecules causes like species to phase separate into different regions of the cell, leading to macromolecular compartmentalization without the need for any intracellular membranes. Strong partitioning and bundling is observed for rodlike biopolymers such as actin or microtubules when mixed with globular proteins or polymers.

The biochemistry of the cell has evolved in this crowded, thermodynamically non-ideal environment, and it may be that the cell has exploited this fact. Prof. Herzfeld [Accounts of Chemical Research 29, 31-37 (1996)] has proposed that that bundling of filaments is driven by macromolecular crowding and that the role of specific bundling proteins is to fine tune certain aspects of the bundling, like the relative alignment of the biopolymers in a bundle. There are a number of questions we are seeking to answer. How crowded do suspensions have to be in order to partition? How strong is the degree of partitioning of the species? What is the organization of the macromolecules in the partitioned phases? How different do the species have to be from each other for this to occur? How does the interparticle potential affect the phenomena? To what extent is all this relevant to cellular biology?

We are studying the partitioning of mixtures of globular and filamentous proteins in vitro using suspensions of the biopolymer fd bacteriophage and Tobacco Mosaic Virus mixed with polymers such as polyethylene glycol and dextran or globular proteins like BSA. Genetic engineering methods are used to systematically alter the length of the biopolymers, an important thermodynamic variable. The viruses are labeled with fluorescent dyes and equilibrated samples are observed in the light microscope. Electron microscopy, light and x-ray scattering, and optical microscopy are used to determine the structure of the macromolecular suspension. We compare our experimental measurements of the phase behavior of these mixtures with several theoretical statistical mechanical models developed by ourselves, by Prof. J. Herzfeld of the Brandeis University Chemistry department and others, as well as with Monte Carlo computer simulations.