Mixed Macromolecular Crowding Accelerates the Oxidative Refolding of Reduced, Denatured Lysozyme

Abstract

The oxidative refolding of reduced, denatured hen egg white lysozyme in the presence of a mixed macromolecular crowding agent containing both bovine serum albumin (BSA) and polysaccharide has been studied from a physiological point of view. When the total concentration of the mixed crowding agent is 100 g/liter, in which the weight ratio of BSA to dextran 70 is 1:9, the refolding yield of lysozyme after refolding for 4 h under this condition increases 24% compared with that in the presence of BSA and 16% compared with dextran 70. A remarkable increase in the refolding yield of lysozyme by a mixed crowding agent containing BSA and Ficoll 70 is also observed. Further folding kinetics analyses show that these two mixed crowding agents accelerate the oxidative refolding of lysozyme remarkably, compared with single crowding agents. These results suggest that the stabilization effects of mixed macromolecular crowding agents are stronger than those of single polysaccharide crowding agents such as dextran 70 and Ficoll 70, whereas the excluded volume effects of mixed macromolecular crowding agents are weaker than those of single protein crowding agents such as BSA. Both the refolding yield and the rate of the oxidative refolding of lysozyme in these two mixed crowded solutions with suitable weight ratios are higher than those in single crowded solutions, indicating that mixed macromolecular crowding agents are more favorable to lysozyme folding and can be used to simulate the intracellular environments more accurately than single crowding agents do.The main question that protein folding encountered in vivo is that the intracellular environment is highly crowded because of the presence of high concentrations of soluble and insoluble macromolecules, which include proteins, nucleic acids, ribosomes, and carbohydrates (polysaccharides) (1-3). It has been estimated that the concentration of macromolecules in cytoplasm is in the range of 80 -400 g/liter (3-5), and all the macromolecules in physiological fluid media collectively occupy a lower limit of ϳ10% and a upper limit of ϳ40% of total fluid volume (1, 2, 6). These media are termed "crowded" or "volumeoccupied" rather than "concentrated" (7,8). The biophysical theory of macromolecular crowding has been well developed by a thermodynamic approach (2, 8 -10). The effect of excluded volume upon protein stability and conformation has been discussed using a simplified statistical thermodynamic model (11). One pertinent prediction of this model is that intramolecular excluded volume will increase the chemical potential of the unfolded state relative to that of the native or compact nonnative state, resulting in a relative stabilization of the native or compact non-native state relative to a fully unfolded state (12).Most of our knowledge about protein folding comes from biophysical studies of the refolding of pure denatured proteins at low concentrations in simple defined buffers carried out in the past three decades since the pioneering studies of Anfinsen (13). However, intra...

Destabilised mutants of ubiquitin gain equal stability in crowded solutions

“…Unlike in other experiments [30,62,20], the rate constant of refolding reached a maximum for all the mutants at around 2000 s −1 . This could represent the maximum refolding rate achievable for ubiquitin under physiological conditions, or it could be a consequence of changes in the denatured state caused by addition of glucose.…”

“…Ubiquitin, like CI2 [20,29], FKBP12 [30,60], lysozyme [22,61,62], ferri-cytochrome C [22,21] and several other proteins increases stability in solutions containing both small osmolytes and larger macromolecules. The equilibrium free energy of unfolding for ubiquitin WT* and the three mutants I13A, K27A and I61V increased by around 1.3±0.4 kcal/mol in the presence of 200 g/L of glucose, a value consistent with other proteins in solutions containing polyols.…”

Macromolecular Crowding and Confinement: Biochemical, Biophysical, and Potential Physiological Consequences

“…It is assumed in these models that crowder molecules interact with each other only via volume exclusion. One indication that the situation in physiological media may be more complex is provided by a recently published report (87), where concentrated mixtures of protein and polysaccharide were shown to exhibit non-additive effects upon the refolding of lysozyme. Moreover, concentrated solution mixtures of dextran and polyethylene glycol have been found to spontaneously separate into immiscible phases, between which proteins may partition in accordance with their relative affinity for each phase (45).…”

The effects of macromolecular crowding on the mechanical stability of protein molecules

“…The persistence length of dextran, determined by single-molecule measurements, was found to be 0.4 nm (at low force regime) (Rief et al 1998), indicating that dextran is a very flexible polymer which should exist as a random coil with a spherical shape in aqueous solutions, especially when present in high concentrations. In previous studies of macromolecular crowding on protein folding stability and kinetics, crowding agents have included dextran (Sasahara et al 2003), Ficoll (Tokuriki et al 2004;Spencer et al 2005), polyethylene glycol (Tokuriki et al 2004;Ai et al 2006), and bovine serum albumin (Qu and Bolen 2002;Zhou et al 2004). …”