Macromolecular crowding is relevant to osmosensing and osmosignaling because crowding can change dramatically as cellular hydration changes in response to osmotic stress, significantly altering the structures, aggregation, and functions of individual macromolecules in the cytoplasm and cytoplasmic membrane (Minton, 2006). The fraction of solution volume occupied by a crowding agent such as a protein or nucleic acid (and hence the volume fraction from which other macromolecules are excluded) can be calculated as
where c is the mass concentration of the crowding agent (grams of solute per milliliter of solution) and v is the partial specific volume of the crowding agent (milliliter of solute per gram of solute). The excluded volume fraction of the non-nucleoid cytoplasm in E. coli has been estimated as 0.3 to 0.4, and such crowding could be simulated by a globular protein with a molecular mass close to 75 kDa at a concentration of 0.34 g/ml (Zhang et al., 1996; Zimmerman and Trach, 1991). Experiments designed to analyze the impact of crowding on macromolecular structure and function require crowding agents that are highly soluble and do not interact (specifically or nonspecifi-cally) with the molecule under study. Bovine serum albumin, cyanomethe-moglobin, and dextran have been used for this purpose (Minton, 1998, 2006; Zimmerman and Minton, 1993).
Large solutes also affect protein structure and aggregation by being sterically excluded from water-filled clefts within or between proteins, hence causing osmotically induced protein dehydration (Parsegian et al., 1995, 2000). Poly(ethylene)glycols (PEGs) are sometimes used as crowding agents and may also act via steric exclusion. Both mechanisms can be probed effectively with PEGs available from Polypure AS (Oslo, Norway; http:// www.polypure.no/) as monodisperse preparations with systematically varying molecular sizes (e.g., Culham et al., 2003). However, PEGs must be used with care because they can interact with proteins (Bhat and Timasheff, 1992); those with molecular masses greater than approximately 3 kDa can catalyze membrane fusion (Lentz and Lee, 1999).
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