Effects of Macromolecular Crowding on the Refolding of Glucose- 6-phosphate Dehydrogenase and Protein Disulfide Isomerase

Abstract

The effects of polysaccharide, polyethylene glycol, and protein-crowding agents on the refolding of glucose-6-phosphate dehydrogenase (G6PDH) and protein disulfide isomerase have been examined. By increasing concentration during refolding, the reactivation yields of the two proteins decrease with the formation of soluble aggregates. In the presence of high concentrations of crowding agents the reactivation yields remain constant but with decreased refolding rates. The refolding of G6PDH changes from monophasic to biphasic first-order reactions in the presence of crowding agents, and the amplitude of the new slow phase increases with increasing concentrations of crowding agents. The molecular chaperone GroEL reverses the refolding kinetics of G6PDH from biphase back to monophase and accelerates the refolding process. Our results display the complexity and diversity of the effects of macromolecular crowding on both the thermodynamics and kinetics of protein folding. The effects of polysaccharide, polyethylene glycol, and protein-crowding agents on the refolding of glucose-6-phosphate dehydrogenase (G6PDH) and protein disulfide isomerase have been examined. By increasing concentration during refolding, the reactivation yields of the two proteins decrease with the formation of soluble aggregates. In the presence of high concentrations of crowding agents the reactivation yields remain constant but with decreased refolding rates. The refolding of G6PDH changes from monophasic to biphasic first-order reactions in the presence of crowding agents, and the amplitude of the new slow phase increases with increasing concentrations of crowding agents. The molecular chaperone GroEL reverses the refolding kinetics of G6PDH from biphase back to monophase and accelerates the refolding process. Our results display the complexity and diversity of the effects of macromolecular crowding on both the thermodynamics and kinetics of protein folding. polyethylene glycol 20000 bovine serum albumin guanidine hydrochloride glucose-6-phosphate dehydrogenase protein disulfide isomerase In the past decades extensive studies on protein folding in vitro have provided a wealth of information for understanding how nascent polypeptides mature into functional proteins in cells and for surmounting difficulties in refolding inclusion bodies of many recombinant proteins. In vitro folding of proteins was performed usually at low concentrations to avoid aggregation during folding; however, in the cytoplasm of cells there are a large number of soluble and insoluble macromolecules at different concentrations, sometimes at very high concentrations depending on physiological and environmental conditions. It has been estimated that the concentration of macromolecules in cytoplasm could be in the range of 80–200 g/liter (1Swaminathan R. Hwang C.P. Verkman A.S. Biophys. J. 1997; 72: 1900-1907Abstract Full Text PDF PubMed Scopus (498) Google Scholar). Therefore all species of macromolecules occupy a large fraction of the total volume of a cell and such a media has been named “crowded” (2Minton A.P. Curr. Opin. Struct. Biol. 2000; 10: 34-39Crossref PubMed Scopus (561) Google Scholar). One of the consequences of crowding is steric repulsion, which derived from the mutual impenetrability of macromolecules, and has been referred to more precisely as volume exclusion (3Zimmerman S.B. Minton A.P. Annu. Rev. Biophys. Biomol. Struct. 1993; 22: 27-65Crossref PubMed Scopus (1292) Google Scholar, 4Minton A.P. J. Biol. Chem. 2001; 276: 10577-10580Abstract Full Text Full Text PDF PubMed Scopus (1216) Google Scholar). This is the most significant difference between the intracellular and artificial in vitroenvironment but has not been considered in the majority of investigations. Very recently Ellis (5Ellis R.J. Curr. Opin. Struct. Biol. 2001; 11: 114-119Crossref PubMed Scopus (896) Google Scholar) suggested that the addition of crowding agents at biologically relevant concentrations to solutions should be considered as a routine variable to meet the objective in studying interactions between biological molecules under more physiologically relevant conditions. Although the biophysical theory of macromolecular crowding by thermodynamic and statistical thermodynamic approaches is well developed (3Zimmerman S.B. Minton A.P. Annu. Rev. Biophys. Biomol. Struct. 1993; 22: 27-65Crossref PubMed Scopus (1292) Google Scholar, 4Minton A.P. J. Biol. Chem. 2001; 276: 10577-10580Abstract Full Text Full Text PDF PubMed Scopus (1216) Google Scholar, 6Minton A.P. Mol. Cell. Biochem. 1983; 55: 119-140Crossref PubMed Scopus (430) Google Scholar, 7Minton A.P. Biophy. J. 1992; 63: 1090-1100Abstract Full Text PDF PubMed Scopus (165) Google Scholar, 8Minton A.P. Colclasure G.C. Parker J.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10504-10506Crossref PubMed Scopus (133) Google Scholar), there are not many biochemical experiments thus far to examine the effects of crowding on protein folding. van den Berg et al. (9van den Berg B. Ellis R.J. Dobson C.M. EMBO J. 1999; 18: 6927-6933Crossref PubMed Scopus (434) Google Scholar) have studied the effect of crowding agents on the refolding of denatured lysozyme. Further kinetic analysis suggested that under conditions resembling the intracellular environment, the rates of many protein-folding processes are likely to be altered (10van den Berg B. Wain R. Dobson C.M. Ellis R.J. EMBO J. 2000; 19: 3870-3875Crossref PubMed Scopus (233) Google Scholar). For chaperone-assisted protein refolding, it has been reported that the release of nonnative polypeptide from GroEL was prevented in the presence of crowding agents (11Martin J. Hartl F.-U. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1107-1112Crossref PubMed Scopus (102) Google Scholar), although contradictory results have also been reported that rhodanese can be released from GroEL in its nonnative form in the milieu of an intact cell (12Burston S.G. Weissman J.S. Farr G.W. Fenton W.A. Horwich A.L. Nature. 1996; 383: 96-99Crossref PubMed Scopus (82) Google Scholar). Recently it has been shown that macromolecular crowding modulates the oligomerization of GroEL during its refolding (13Galán A. Sot B. Llorca O. Carrascosa J.L. Valpuesta J.M. Muga A. J. Biol. Chem. 2001; 276: 957-964Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In this communication we have studied the effects of polysaccharide (Ficoll 70 and Dextran 70), polyethylene glycol 20000 (PEG),1 and the protein crowding agents bovine serum albumin (BSA), chicken egg ovalbumin, and lysozyme on the refolding of guanidine hydrochloride (GdnHCl)-denatured glucose-6-phosphate dehydrogenase (G6PDH) and protein disulfide isomerase (PDI) at 25 and 37 °C. The results show that the renaturation yields of the two proteins do not change in the presence of high concentrations (50–200 g/liter) of crowding agents; however, the refolding kinetics change markedly. The chaperone GroEL promotes the folding and reverses the effects of crowding agents on G6PDH folding. G6PDH (type V, from baker yeast), BSA, ovalbumin, and GdnHCl were purchased from Sigma, PEG and chicken egg lysozyme were purchased from Roche Molecular Biochemicals, and Ficoll 70 and Dextran 70 were purchased from Amersham Pharmacia Biotech. PDI was prepared from bovine liver, and the thiol-protein oxidoreductase activity was assayed according to Lambert and Freedman (14Lambert N. Freedman R.B. Biochem. J. 1983; 213: 225-234Crossref PubMed Scopus (130) Google Scholar). Escherichia coli GroEL was prepared according to Li and Wang (15Li J. Wang C.C. J. Biol. Chem. 1999; 274: 10790-10794Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). G6PDH was assayed according to Hansen and Gafni (16Hansen J.E. Gafni A. J. Biol. Chem. 1993; 268: 21632-21636Abstract Full Text PDF PubMed Google Scholar). In all experiments, 0.1m potassium phosphate buffer (pH 7.5) containing 2.5 mm EDTA was used and referred to as phosphate buffer. The concentrations of G6PDH and PDI were determined spectrophotometrically at 280 nm with the absorption coefficient (A 1 cm0.1% ) of 1.15 for G6PDH (16Hansen J.E. Gafni A. J. Biol. Chem. 1993; 268: 21632-21636Abstract Full Text PDF PubMed Google Scholar) and 0.9 for PDI (17Hu C.-H. Wang C.-C. Chin. Biochem. J. 1988; 4: 61-66Google Scholar). The concentration of GroEL was determined by the method of Bradford (18Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (222415) Google Scholar) with BSA as a standard. G6PDH and PDI were taken as monomers and GroEL as a tetradecamer in the calculations of concentrations, because oligomeric G6PDH and PDI were presumably dissociated to monomers when fully denatured. Denaturation of G6PDH was carried out by incubation of 12 or 24 μm enzyme with 4 m GdnHCl at 25 °C for 24 h (16Hansen J.E. Gafni A. J. Biol. Chem. 1993; 268: 21632-21636Abstract Full Text PDF PubMed Google Scholar). PDI at 140 or 280 μm was fully denatured by treatment with 6.4m GdnHCl at 25 °C for 24 h (19Morjana N.A. McKeone B.J. Gilbert H.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2107-2111Crossref PubMed Scopus (79) Google Scholar). The refolding of denatured G6PDH or PDI was initiated by quick dilution at least 15-fold in phosphate buffer containing different concentrations of crowding agents and/or chaperone GroEL at 25 or 37 °C. In addition to PEG and polysaccharides (Ficoll 70 and Dextran 70) as background species, BSA, ovalbumin, and lysozyme were used as protein-crowding agents. The presence of the above crowding agents from 50 to 200 g/liter was to mimic the intracellular crowded environment in vitro. GroEL to G6PDH molar ratio of 2 has been determined to help the refolding of the substrate to a maximal level (16Hansen J.E. Gafni A. J. Biol. Chem. 1993; 268: 21632-21636Abstract Full Text PDF PubMed Google Scholar). The refolding of G6PDH or PDI in the absence of crowding agents takes about 1 h to reach maximal activity recovery, which does not change in 24 h. The activity recovery was thus determined in 24 h after dilution, and the reactivation yield was expressed as a percentage of the activity of native enzyme, which was incubated at 25 or 37 °C for at least 24 h and showed full activity. The aggregation during refolding was monitored by 90° light scattering at 488 nm continuously in a Shimadzu RF5301PC spectrofluorometer. The residual denaturants in the refolding system had been examined and determined to have no effect on the activities of the enzymes and the chaperone activity of GroEL. Denatured G6PDH (12 μm) and denatured PDI (140 μm) was refolded by dilution to 0.36 and 7 μm, respectively, at 25 °C. The protein concentrations in the supernatants in refolding products after at for were by at 280 the supernatants in the refolding products were in 24 h a in a with a of The of the reactivation of G6PDH and PDI in the absence or presence of crowding agents and/or were determined at 25 and 37 °C. The were well to for first-order reactions in monophasic in biphasic shown in 1 the reactivation yield of G6PDH from to at 25 °C and to at 37 °C with the concentration of denatured protein increasing from to The reactivation yield of PDI from to at 25 °C and to at 37 °C when the concentration increases from to 1 The concentration of the refolding of the two proteins at 37 °C more significant that at 25 °C. with the decrease in the reactivation aggregation during refolding increases with increasing protein concentrations at both of 25 and 37 °C the reactivation yields at 25 °C are at 37 °C at all protein concentrations most of the experiments were carried out at 25 °C. G6PDH μm) and PDI μm) were used to examine the effect of crowding agents on the refolding for aggregation is by light scattering during the refolding of G6PDH μm) and PDI μm) at 25 or 37 although the reactivation yields of the two enzymes are far from By the analysis shown in native G6PDH as a with an molecular of and PDI as a of with a as a of The in refolding of G6PDH in the volume with an molecular The ratio of high molecular to the at the of the native enzyme is of also in the in refolding of PDI with a ratio to the refolded molecules of The above results that the oligomeric folding products during refolding be very and the molecular of most are not large to be by the light scattering the soluble proteins in the in the refolding products of G6PDH and PDI are and of all proteins by the at 280 respectively, it is to be that the of the at the of native proteins in the total refolding for G6PDH and for are to the reactivation yield of and respectively, for G6PDH and It that the oligomeric refolding although most are are and for the decreased activity BSA, ovalbumin, and but not Ficoll 70 and Dextran were as crowding agents for G6PDH refolding, because are on G6PDH activity after incubation at 200 g/liter with native G6PDH at 25 and 37 °C for 24 h. shown in the reactivation yields of G6PDH in the presence of the crowding agents to 200 g/liter do not change at 25 and 37 °C. Ficoll Dextran and PEG were as crowding agents for PDI refolding because do not the activity of and with the PDI BSA, ovalbumin, and lysozyme could not be because the disulfide in proteins at high concentrations with the activity of to the reactivation of the crowding agents at increasing concentrations to 200 g/liter do not the reactivation yield of denatured PDI at 25 and 37 °C The of G6PDH refolding at 25 and 37 °C in the absence of crowding agents are well to monophasic first-order kinetics with the kinetic shown in The presence of BSA does not change the first-order of the kinetics but the folding BSA concentration to 200 the refolding with refolding in 4 and and the presence of 4 and lysozyme or PEG 4 the refolding kinetics of G6PDH to biphasic first-order reactions with the as phase and the as slow phase 25 °C with the concentration of and lysozyme to 200 the amplitude of the to with a decrease of at the of the slow decreased The effects of PEG are more with decreased to and decreased to when the concentration increases to 200 however, change of G6PDH refolding under crowding conditions at 37 °C. Denatured G6PDH was refolded at 0.36 μm in the absence or presence of 200 g/liter BSA or The are expressed as of G6PDH refolding in the presence of crowding were carried out at 25 and 37 °C as in the to 4 and are expressed as in a new were carried out at 25 and 37 °C as in the to 4 and are expressed as The refolding of PDI to monophasic first-order kinetics in the presence of 200 g/liter crowding agents at both 25 and 37 °C with the decreased to and for Ficoll 70 and to for Dextran 70 The for the refolding of G6PDH and PDI are at 37 °C at 25 °C in the absence or presence of crowding agents with 4 and the presence of increases the reactivation yields of G6PDH from to at 25 °C and from to at 37 °C in the presence of 200 g/liter BSA, ovalbumin, or which the at both 7 and GroEL reverses the folding kinetics of G6PDH under crowding conditions from biphasic to monophasic first-order reactions and the folding from 2 to 1 h in the presence of 200 g/liter BSA and from 24 to 2 h with 200 g/liter and lysozyme. The above that crowding agents do not the refolding yields of G6PDH but do slow the folding on the GroEL to the effect of crowding agents on G6PDH refolding In to the absence of the refolding at 37 °C the at 25 °C of refolding of G6PDH in the presence of crowding are carried out as in the to are expressed as in a new are carried out as in the to are expressed as to the crowding macromolecules, by volume to the and the of a and the of species of in the (2Minton A.P. Curr. Opin. Struct. Biol. 2000; 10: 34-39Crossref PubMed Scopus (561) Google Scholar). This results in the of or products and the most of the which the least volume to the background reactions are under crowding conditions with the which could be of in solutions (3Zimmerman S.B. Minton A.P. Annu. Rev. Biophys. Biomol. Struct. 1993; 22: 27-65Crossref PubMed Scopus (1292) Google Scholar, 4Minton A.P. J. Biol. Chem. 2001; 276: 10577-10580Abstract Full Text Full Text PDF PubMed Scopus (1216) Google Scholar). In this van den Berg et al. (9van den Berg B. Ellis R.J. Dobson C.M. EMBO J. 1999; 18: 6927-6933Crossref PubMed Scopus (434) Google Scholar) have reported that high concentrations of BSA, ovalbumin, Ficoll and Dextran 70 the refolding of and denatured lysozyme because of the aggregation and the refolding (10van den Berg B. Wain R. Dobson C.M. Ellis R.J. EMBO J. 2000; 19: 3870-3875Crossref PubMed Scopus (233) Google Scholar). have that results are with the of volume theory (9van den Berg B. Ellis R.J. Dobson C.M. EMBO J. 1999; 18: 6927-6933Crossref PubMed Scopus (434) Google Scholar). The we in the on the refolding of G6PDH and PDI in the presence of crowding agents have shown a different G6PDH from J. Biol. Chem. Full Text PDF PubMed Google Scholar) and PDI from bovine are both molecules under the conditions of this a of PDI as a The reactivation yields of G6PDH and depending on the protein concentration and are far from under the conditions The the protein concentration and the the the reactivation yields and the the during the refolding of G6PDH at 0.36 μm and PDI at 7 μm can be by light scattering or by at at 25 and of by analysis form and to and The above that to that for which form or during aggregation during the folding of G6PDH or although is also the with folding and is for the the of two is for the reactivation of denatured G6PDH or PDI in the refolding kinetics are shown in In folding two or refolded of G6PDH or PDI to a folding on the change from a to the native In aggregation to with the folding of and has been suggested to the folding of proteins that are to aggregation (9van den Berg B. Ellis R.J. Dobson C.M. EMBO J. 1999; 18: 6927-6933Crossref PubMed Scopus (434) Google Scholar) but not to this (5Ellis R.J. Curr. Opin. Struct. Biol. 2001; 11: 114-119Crossref PubMed Scopus (896) Google Scholar). from a protein that is to G6PDH and PDI are both molecules with a to change of the refolding yields of G6PDH and PDI in a crowding environment with 200 g/liter crowding agents from the of the two effects of to the of for the of molecules and to the although not The between the above two is in crowded conditions. In this it has been reported that crowding modulates the oligomerization of GroEL in two different it increases the of of monomers at and the rates of the two the to native GroEL and aggregation (13Galán A. Sot B. Llorca O. Carrascosa J.L. Valpuesta J.M. Muga A. J. Biol. Chem. 2001; 276: 957-964Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Although crowding agents the aggregation of it its The effects of crowding on the folding of a protein on the of aggregation and of the and the effects between shown in is a and on the of two is a first-order depending on It has been reported that the reactivation kinetics of G6PDH at concentrations μm a first-order and the change from an folding to native form is a Gafni A. Biophys. 1993; Scopus Google Scholar). The effects of crowding on and change with to rates (5Ellis R.J. Curr. Opin. Struct. Biol. 2001; 11: 114-119Crossref PubMed Scopus (896) Google Scholar). In the maximal for has been suggested to be when the concentration of crowding agents high (3Zimmerman S.B. Minton A.P. Annu. Rev. Biophys. Biomol. Struct. 1993; 22: 27-65Crossref PubMed Scopus (1292) Google Scholar, 4Minton A.P. J. Biol. Chem. 2001; 276: 10577-10580Abstract Full Text Full Text PDF PubMed Scopus (1216) Google Scholar, R.J. Curr. Opin. Struct. Biol. 2001; 11: 114-119Crossref PubMed Scopus (896) Google Scholar). it is that the refolding kinetics of G6PDH and PDI show the of a first-order in or crowded that change but not is the the crowding the refolding of oligomeric G6PDH and PDI but increases the refolding of lysozyme (9van den Berg B. Ellis R.J. Dobson C.M. EMBO J. 1999; 18: 6927-6933Crossref PubMed Scopus (434) Google Scholar). In to the volume we the folding in the presence of crowding agents to the The folding of to an which results in and the change during the is the which is with the decreased refolding rates under crowding conditions (2Minton A.P. Curr. Opin. Struct. Biol. 2000; 10: 34-39Crossref PubMed Scopus (561) Google Scholar). It has been that in the refolding of bovine two kinetic with of and R.J. Curr. Opin. Struct. Biol. 2001; 11: 114-119Crossref PubMed Scopus (896) Google Scholar, 6Minton A.P. Mol. Cell. Biochem. 1983; 55: 119-140Crossref PubMed Scopus (430) Google Scholar, 7Minton A.P. Biophy. J. 1992; 63: 1090-1100Abstract Full Text PDF PubMed Scopus (165) Google Scholar, 8Minton A.P. Colclasure G.C. Parker J.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10504-10506Crossref PubMed Scopus (133) Google Scholar, den Berg B. Ellis R.J. Dobson C.M. EMBO J. 1999; 18: 6927-6933Crossref PubMed Scopus (434) Google Scholar, den Berg B. Wain R. Dobson C.M. Ellis R.J. EMBO J. 2000; 19: 3870-3875Crossref PubMed Scopus (233) Google Scholar, J. Hartl F.-U. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1107-1112Crossref PubMed Scopus (102) Google Scholar, S.G. Weissman J.S. Farr G.W. Fenton W.A. Horwich A.L. Nature. 1996; 383: 96-99Crossref PubMed Scopus (82) Google Scholar, A. Sot B. Llorca O. Carrascosa J.L. Valpuesta J.M. Muga A. J. Biol. Chem. 2001; 276: 957-964Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, N. Freedman R.B. Biochem. J. 1983; 213: 225-234Crossref PubMed Scopus (130) Google Scholar) to a to with as an the fully native with of Biol. Chem. 1997; Google Scholar). The refolding of rhodanese was also in the presence of but with a refolding yield with that in the absence of the (12Burston S.G. Weissman J.S. Farr G.W. Fenton W.A. Horwich A.L. Nature. 1996; 383: 96-99Crossref PubMed Scopus (82) Google Scholar). In it has been that the effect of crowding on is and on the of the the species, and the concentrations of crowding agents (5Ellis R.J. Curr. Opin. Struct. Biol. 2001; 11: 114-119Crossref PubMed Scopus (896) Google Scholar). The most of the effect of crowding agents on the refolding of G6PDH is to change the refolding kinetics from monophasic to biphasic of the first-order increasing the concentration of crowding agents, the amplitude of the slow phase increases and that of the phase but with decreased for both for G6PDH refolding that the presence of crowding results in a new folding with more and the be that and results in a folding with increasing concentrations of crowding agents, G6PDH folding in the more as to the amplitude of the slow BSA is an crowding and at high concentrations the refolding kinetics of G6PDH It has been that BSA the refolding of denatured and lysozyme also from the crowding agents and has been suggested for this effect of BSA (9van den Berg B. Ellis R.J. Dobson C.M. EMBO J. 1999; 18: 6927-6933Crossref PubMed Scopus (434) Google Scholar). The absence of large effects of crowding on the refolding of denatured and lysozyme was suggested to be by the high of this with the refolding of denatured and the refolding of which was under crowding conditions (9van den Berg B. Ellis R.J. Dobson C.M. EMBO J. 1999; 18: 6927-6933Crossref PubMed Scopus (434) Google Scholar). It was suggested that proteins more in as a of to aggregation (9van den Berg B. Ellis R.J. Dobson C.M. EMBO J. 1999; 18: 6927-6933Crossref PubMed Scopus (434) Google Scholar). In the refolding yield of lysozyme in the presence of crowding agents decreased because of aggregation the refolding was (9van den Berg B. Ellis R.J. Dobson C.M. EMBO J. 1999; 18: 6927-6933Crossref PubMed Scopus (434) Google Scholar). The refolding yields of G6PDH and PDI do not change but have folding rates under crowding conditions. It that effects of crowding on the folding and aggregation of different proteins could be One of the of cells to the effects of crowding is to molecular as products R.J. Curr. Biol. 1997; Full Text Full Text PDF PubMed Google Scholar). the refolding yield of and crowding is not to with the effect of chaperone GroEL but the folding because the of the be prevented by the crowding agents as to substrate folding. BSA and Ficoll decrease the refolding of lysozyme (9van den Berg B. Ellis R.J. Dobson C.M. EMBO J. 1999; 18: 6927-6933Crossref PubMed Scopus (434) Google Scholar). The effects of macromolecular crowding on chaperone-assisted refolding of different proteins also The most effect of on the refolding of G6PDH under crowding conditions in the is that GroEL the refolding kinetics from biphasic to monophasic with the of the slow phase by crowding agents and thus accelerates the refolding with the refolding under crowding conditions. the effects of crowding and to the folding of proteins as a G6PDH refolding at 37 °C more but at 25 between G6PDH and GroEL at a (16Hansen J.E. Gafni A. J. Biol. Chem. 1993; 268: 21632-21636Abstract Full Text PDF PubMed Google Scholar). It has been reported that the between GroEL and denatured by increasing the in the and the reactivation as an A. J. Mol. Biol. PubMed Scopus Google Scholar). The effects of crowding agents on the refolding of G6PDH or PDI are at 25 and 37 °C the refolding kinetics of lysozyme in the presence of macromolecular crowding was studied at °C (10van den Berg B. Wain R. Dobson C.M. Ellis R.J. EMBO J. 2000; 19: 3870-3875Crossref PubMed Scopus (233) Google Scholar) but not at 37 °C for studies on refolding and aggregation (9van den Berg B. Ellis R.J. Dobson C.M. EMBO J. 1999; 18: 6927-6933Crossref PubMed Scopus (434) Google Scholar). The macromolecular crowding is but it is a of the intracellular environment (5Ellis R.J. Curr. Opin. Struct. Biol. 2001; 11: 114-119Crossref PubMed Scopus (896) Google Scholar). have the refolding of G6PDH and PDI under crowding conditions and that folding yields could be at the of the decrease of folding rates in crowded cells for and molecular can the effects of with the refolding of lysozyme under crowding it that the volume effect on protein folding is very and and on the of and crowding agents.