Macromolecular Crowding

INTRODUCTION Protein folding is a process by which a polypeptide chain folds into its native three dimensional structure, a conformation that is biologically functional. It is most often assumed that protein folding and its biophysical and structural properties observed in dilute buffer solutions in vitro also represent the in vivo scenario. However the intracellular environment is highly crowded because of the presence of large amounts of soluble and insoluble biomolecules including proteins, nucleic acids, osmolytes, ribosomes and carbohydrates. [reference] It has been estimated that the concentration of macromolecules in the cytoplasm ranges from 80 to 400 mg/ml [life in crowded world, rivas, 2004]. All macromolecules in physiological fluids

This means that a significant fraction of the intracellular space is not available to other macromolecular species. The term “macromolecular crowding”, coined by Minton [minton,1981, effect of mc upon the structure and function of an enzyme], implies the nonspecific influence of steric repulsions on specific reactions that occur in highly volume-occupied media. Crowding results in “volume exclusion”, specific as well as non-specific intermolecular interaction and increased viscosity as compared to in dilute solutions. Wheras change in viscosity should not affect thermodynamics but excluded volume effect and interaction will affect protein equilibrium states. One of the areas wherein macromolecular crowding has made appreciable impact is protein structure, function and stability. [reference] Experimental and theoretical work have demonstrated large effects of macromolecular crowding on the thermodynamics and kinetics of many biological processes. The influence of macromolecules on protein stability is thought to arise from two phenomena: hard-core repulsion and chemical interactions. The repulsive interaction is always stabilizing because it involves only the arrangement of molecules and they affect the entropic component of

The MG state is a compact denatured state with a significant native like secondary structure but a largely disordered tertiary structure. In addition, there are studies demonstrating that proteins can convert from unfolded to folded or molten-globule states upon addition of large amounts of crowding agents. For instance, unfolded cytochrome c at pH 2 can adopt a molten globule structure in the presence of crowding agents, unfolded RNase A at pH 3 adopts a folded-like structure upon addition of 350 mg/ml PEG 20,000 or Ficoll 70, and the reduced and carboxyamidated form of RNase T1 that is intrinsically unstructured at pH 7 was found to exhibit some catalytical activity upon the addition of 400 mg/ml dextran 70. In addition, protein binding to a membrane surface results in “partial denaturation” (i.e. being transformed into a non-native state). The effects of various polyols, such as ethylene glycol, glycerol, erythritol, xylitol, sorbitol, and inositol, on the structure of acid-unfolded horse cytochrome c at pH 2 were investigated. The addition of polyols induces the characteristic CD spectra of the molten globule. Davis-Searles et al. have recently reported that sugars induce the molten globule state of cytochrome c. This is mainly due to the