Mixed Macromolecular Crowding Accelerates the Refolding of Rabbit Muscle Creatine Kinase: Implications for Protein Folding in Physiological Environments

doi:10.1016/j.jmb.2006.09.018

J. Mol. Biol. (2006) 364, 469–482

Mixed Macromolecular Crowding Accelerates the Refolding of Rabbit Muscle Creatine Kinase: Implications for Protein Folding in Physiological Environments Fen Du, Zheng Zhou, Zhong-Ying Mo, Jun-Zhi Shi, Jie Chen and Yi Liang⁎ State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China

The effects of four single macromolecular crowding agents, Ficoll 70, dextran 70, polyethylene glycol (PEG) 2000, and calf thymus DNA (CT DNA), and three mixed crowding agents containing both CT DNA and polysaccharide (or PEG 2000) on the refolding of guanidine hydrochloridedenatured rabbit muscle creatine kinase (MM-CK) have been examined by activity assay. When the total concentration of the mixed crowding agent is 100 g/l, in which the weight ratio of CT DNA to Ficoll 70 is 1:9, the refolding yield of MM-CK after refolding for 3 h under these conditions increases 23% compared with that in the presence of 10 g/l CT DNA, 18% compared with 100 g/l Ficoll 70, and 19% compared with that in the absence of crowding agents. A remarkable increase in the refolding yield of MM-CK by a mixed crowding agent containing CT DNA and dextran 70 (or PEG 2000) is also observed. Further folding kinetics analyses show that these three mixed crowding agents remarkably accelerate the refolding of MM-CK, compared with single crowding agents. Aggregation of MM-CK in the presence of any of the three mixed crowding agents is less serious than that in the presence of a single crowding agent at the same concentration but more serious than that in the absence of crowding agents. Both the refolding yield and the refolding rate of MM-CK in mixtures of these agents are increased relative to the individual agents by themselves, indicating that mixed macromolecular crowding agents are more favorable to MM-CK folding and can be used to reflect the physiological environment more accurately than single crowding agents. © 2006 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: creatine kinase; macromolecular crowding; protein refolding; folding kinetics; protein aggregation

Introduction Protein refolding in vitro has been extensively characterized under dilute experimental conditions Abbreviations used: CK, creatine kinase; MM-CK, cytosolic creatine kinase isoenzyme from rabbit muscle; Cr, creatine; PCr, phosphocreatine; CT DNA, calf thymus DNA; GAPDH, D-glyceraldehyde-3-phosphate dehydrogenase; GdnHCl, guanidine hydrochloride; UV-vis, ultraviolet and visible; DFT, density-functional theory. E-mail address of the corresponding author: [email protected]

to avoid aggregation of unstable intermediates. However, the intracellular environment is, in fact, highly crowded due to the presence of high concentrations of soluble and insoluble macromolecules, which include proteins, nucleic acids, ribosomes, and carbohydrates (polysaccharides), so that a significant fraction of the intracellular space is not available to other macromolecular species.1–5 It has been estimated that the concentration of macromolecules in cytoplasm is in the range of 80 g/l– 400 g/l6–8 and all the macromolecules in physiological fluid media collectively occupy a lower limit of about 10% and a upper limit of about 40% of total fluid volume.9,10 In Escherichia coli, the average DNA concentration within the nucleoid is estimated

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470 as 30–100 g/l, similar to the concentration of DNA in the interphase nucleus of eukaryotic cell.11 These media are termed “crowded” or “volume-occupied” rather than “concentrated”.12,13 Any reactions that increase the available volume are predicted to be stimulated by macromolecular crowded conditions. These processes include the binding of macromolecules to one another, the folding of protein and nucleic acid chains into more compact shapes, and the formation of aggregates, such as the amyloid deposits seen in some neurodegenerative diseases.2 Given the complexity of the intracellular milieu, the effects of macromolecular crowding on the function of retrovirus integrase have also been examined.14,15 Another effect of macromolecular crowding is to reduce the rate of diffusion by factors up to 10, depending on the size of the diffusing particle and the degree of occupancy of the medium, compared with the rate in uncrowded solutions. Thus, a biochemical reaction is influenced by macromolecular crowding if its rate is limited by diffusion or by the stability of macromolecular complexes.2 The biophysical theory of macromolecular crowding has been well developed by a thermodynamic approach.16 The excluded volume theory has been well studied and has predicted that many estimates of reaction rates and equilibria made with uncrowded solutions in the test tube differ from those of the same reactions carried out under crowded conditions by orders of magnitude.13,14,17,18 Cheung et al. have used an off-lattice model of the all-β-sheet WW domain in the presence of large spherical particles whose interaction with the polypeptide chain is purely repulsive to study the effects of macromolecular crowding on folding thermodynamics and kinetics of globular proteins.19 Very recently, Minton has presented statistical-thermodynamic models to calculate the excluded volume interaction between an unfolded protein and a polymer modeled as a long rigid rod.20 But there are not many biochemical experiments so far to examine the effects of macromolecular crowding on protein folding. Because macromolecular crowding is an important but neglected aspect of the intracellular environment, more researchers have studied the mechanism of protein refolding under crowding conditions. Minton et al. have studied the specific activity of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in high concentrations of ribonuclease A, β-lactoglobulin, bovine serum albumin, and polyethylene glycol (PEG), and have found that the addition of high concentrations of unrelated globular proteins do not affect the activity of either monomer or tetramer but promote the formation of tetramer due to space-filling properties of the added species.21 The effects of crowding agents on the refolding of reduced, denatured lysozyme,3,22,23 the oligomerization of GroEL subunits, 24 the noncooperative self-assembly of bacterial cell division protein FtsZ,25 the amyloid formation of human apolipoprotein C-II,26 and the self-assembly of capsid protein of human immunodeficiency virus

Mixed Crowding Accelerates MM-CK Refolding

type 127 have been reported in recent years. It has also been reported that the molecular chaperone GroEL changes the refolding kinetics of glucose-6phosphate dehydrogenase from biphasic under macromolecular crowding conditions back to monophasic and accelerates the refolding process.28 The effects of crowding agents on the denaturation of homotetrameric GAPDH in 0.5 M guanidine hydrochloride (GdnHCl) and the reactivation of the fully denatured enzyme have also been examined quantitatively.29 Furthermore, the thermal stability of hen egg-white lysozyme and the conformational change of cytochrome c in the unfolded state have been investigated over an extended concentration range of dextran that can mimic the effect of intracellular crowding on protein stabilization.30 van den Berg et al.22,23 have studied extensively the effects of macromolecular crowding agents on the refolding of reduced, denatured lysozyme and have demonstrated that the correct refolding of lysozyme is essentially abolished due to aggregation at high concentration of crowding agents in the absence of urea. However, the presence of high concentrations of macromolecular crowding agents results in the acceleration of the fast track of the refolding process whereas the slow track is substantially retarded in the presence of 2 M urea.22,23 Recently, we have demonstrated that both the refolding yield and the rate of the oxidative refolding of lysozyme in mixed crowded solutions with suitable weight ratios are higher than those in single crowded solutions, indicating 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.3 Creatine kinase (CK, EC 2.7.3.2) is a key enzyme for energy homeostasis in cells and plays a significant role in the transport of high-energy phosphates via phosphocreatine (PCr) to sites of ATP utilization in vivo.31–34 This enzyme catalyzes the reversible phosphoryl transfer between ATP and creatine (Cr) in the presence of Mg2+, and the release of an equimolar quantity of hydrogen ion. As most of CK in the body normally exists in muscle, the elevated level of CK in human blood is an important diagnostic indicator for diseases of the nervous system and the heart muscle, for malignant hypothermia and for certain tumors. CK molecules also locate and function in certain highly specialized non-muscle tissues and cells, such as retina photoreceptor cells, brain cells, kidney, pancreas, thymus, thyroid, intestinal brush-border epithelial cells, cartilage and bone cells, macrophages, tumor, and cancer cells. 35 Cytosolic creatine kinase from rabbit muscle (MM-CK) is a dimer of two identical 43 kDa subunits of known sequence.36 The crystal structure of the enzyme at 2.35 Å resolution has revealed that the dimeric interface of the enzyme is held together by a small number of hydrogen bonds.37 During the past decade, effects of macromolecular crowding on the folding of several proteins have

Mixed Crowding Accelerates MM-CK Refolding

been extensively studied as described above, but it seems that the excluded volume effect on protein folding is very complex and diverse and depends on the nature of proteins, interactions, and crowding agents.28 Although crowding theory makes simple predictions about the effects of crowding agents on refolding rates,13,14,17,18 conditions in the cell are complex and the effects of intracellular conditions on rates are far from being understood. In order to mimic conditions in the cell to some extent and establish the concept “mixed macromolecular crowding” further, we added both nucleic acid crowding agent and polysaccharide (or polyethylene glycol, PEG) to the refolding buffer, and tested the effect of such mixed macromolecular crowding on protein folding from a physiological point of view. In intracellular environments nucleic acids are at high concentrations, but RNA is unstable in vitro, so we chose calf thymus DNA (CT DNA) as a nucleic acid crowding agent. The mechanistic basis of the effect of mixtures is worth thinking about, because certainly the inside of a cell contains both protein and DNA. In this study, the refolding of GdnHCl-denatured MM-CK, a well-studied dimeric protein, in the presence of such mixed macromolecular crowding agents was investigated. The experimental results indicate that mixed macromolecular crowding agents are more favorable to MM-CK folding and can be used to reflect the physiological environment more accurately than individual crowding agents.

471 experiments assayed after 24 h showed that enzymatic activities of the refolding samples increased only slightly after 3 h of refolding. As shown in Figure 1, in the absence of crowding agents, the recovery of enzymatic activity was biphasic, reaching a maximum at 30 min, and about 68% of the active dimeric form of MM-CK was recovered after refolding for 3 h. As shown in Figures 1–4, the effects of single crowding agents on the refolding of MM-CK were diverse and depended on the nature of the crowding agents used. In the presence of high concentrations of Ficoll 70 (up to 200 g/l) or CT DNA (up to 20 g/l), the reactivation yield of MM-CK almost did not change, but in the presence of high concentrations of dextran 70 and PEG 2000, both up to 200 g/l,

Results Effects of single macromolecular crowding agents on MM-CK refolding The effect of enzyme concentration on the time course of reactivation of MM-CK has been studied extensively.38,39 The results have shown that, within the concentration range studied (0.4 μM −2.6 μM), the process of reactivation of MM-CK is independent of enzyme concentration.38,39 The concentration of MM-CK used here was 1.2 μM, in the same range as described above. The concentrations of crowding agents used here were in the same range as macromolecular concentrations found in the cytoplasm.8,11 The advantage of using polysaccharide crowding agents, such as dextran 70 and Ficoll 70, is that they are inert, polar and do not interfere with some spectroscopic experiments. Protein crowding agents such as bovine serum albumin (BSA) and nucleic acid crowding agents such as CT DNA are more relevant than polysaccharide crowding agents from a physiological point of view, but BSA at high concentration will seriously disturb the measurement of both the activity assay and the intrinsic fluorescence of MM-CK. Therefore, dextran 70, Ficoll 70, CT DNA, and PEG 2000, which had no influence on the activity assay, were employed as single crowding agents in this study. Control

Figure 1. Effect of dextran 70 on the reactivation of denatured MM-CK. (a) Yield of MM-CK refolding in the absence of crowding agents (white bar) and in the presence of 100 g/l dextran 70 (gray bar) or 200 g/l dextran 70 (black bar) after refolding for 3 h. (b) Refolding kinetics of MM-CK in the absence of crowding agents (filled squares) and in the presence of 100 g/l dextran 70 (open circles) or 200 g/l dextran 70 (filled triangles). Denatured MM-CK (1.2 μM) was refolded at pH 7.5 and 25 °C. All enzymatic activities were normalized against the enzymatic activity of 1.2 μM native MM-CK solution in the refolding buffer. The data were fitted to a double exponential model, and the correlation constants for the fittings were 0.9994 (filled square), 0.9995 (open circles), and 0.9988 (filled triangle), respectively. The data with error bars are expressed as the mean ± S.D. (n = 3–5).

472

Mixed Crowding Accelerates MM-CK Refolding

parameters of the refolding of MM-CK were determined. As can be seen from Figures 1(b), 2(b), 3(b), 4(b), the refolding of MM-CK followed the biphasic first-order mechanism in the presence of single macromolecular crowding agents, but the time when the refolding MM-CK reached its maximum activity in the presence of single crowding agents became much longer than that in the absence of crowding agents. Table 1 summarizes the kinetic parameters obtained for the refolding of denatured MM-CK in the presence of single crowding agents. As shown in Table 1, the rate for the fast track of the refolding of MM-CK in the presence of these single crowding agents was 8.2 to 22-fold decreased compared with that in the absence of crowding agents. In contrast, the rate for the slow track of the refolding of MM-CK in the presence of these single crowding agents was 2.3 to 19-fold increased compared with that in the absence of

Figure 2. Effect of Ficoll 70 on the reactivation of denatured MM-CK. (a) Yield of MM-CK refolding in the absence of crowding agents (white bar) and in the presence of 100 g/l Ficoll 70 (gray bar) or 200 g/l Ficoll 70 (black bar) after refolding for 3 h. (b) Refolding kinetics of MM-CK in the absence of crowding agents (filled squares) and in the presence of 100 g/l Ficoll 70 (open circles) or 200 g/l Ficoll 70 (filled triangles). The data were fitted to a double exponential model, and the correlation constants for the fittings were 0.9994 (filled squares), 0.9990 (open circles), and 0.9996 (filled triangles), respectively. The data with error bars are expressed as the mean ± S.D. (n = 3–5).

the refolding yield remarkably decreased from 68% to 46.5%, and 31%, respectively. A remarkable decrease in the refolding yield of MM-CK by PEG 2000 at 100 g/l was also observed (Figure 3). As shown in Table 1, noticeable decreases in the total maximal refolding yield of denatured MMCK (Ymax,1 + Ymax,2) by dextran 70 or PEG 2000 at 200 g/l were also observed. In contrast, only slight changes in the total maximal refolding yield caused by high concentrations of Ficoll 70 or CT DNA were observed. As shown in Figures 1–4 and Table 1, the total maximal refolding yield of MM-CK in the presence of single crowding agents almost accorded with the final refolding yield measured. The difference of refolding kinetics among these single crowding agents was observed clearly as shown in Figures 1–4, from which the kinetic

Figure 3. Effect of PEG 2000 on the reactivation of denatured MM-CK. (a) Yield of MM-CK refolding in the absence of crowding agents (white bar) and in the presence of 100 g/l PEG 2000 (gray bar) or 200 g/l PEG 2000 (black bar) after refolding for 3 h. (b) Refolding kinetics of MM-CK in the absence of crowding agents (filled squares) and in the presence of 100 g/l PEG 2000 (open circles) or 200 g/l PEG 2000 (filled triangles). The data were fitted to a double exponential model, and the correlation constants for the fittings were 0.9994 (filled squares), 0.9972 (open circles), and 0.9957 (filled triangles), respectively. The data with error bars are expressed as the mean ± S.D. (n = 3–5).

473

Mixed Crowding Accelerates MM-CK Refolding

slow track of the refolding, compared with no crowding agent. From Table 1, it can also be seen that the concentration of CT DNA as a crowding agent was one order of magnitude lower than those of other single crowding agents used. Effects of mixed macromolecular crowding agents on refolding yield of MM-CK

Figure 4. Effect of CT DNA on the reactivation of denatured MM-CK. (a) Yield of MM-CK refolding in the absence of crowding agents (white bar) and in the presence of 10 g/l CT DNA (gray bar) or 20 g/l CT DNA (black bar) after refolding for 3 h. (b) Refolding kinetics of MM-CK in the absence of crowding agents (filled squares) and in the presence of 10 g/l CT DNA (open circles) or 20 g/l CT DNA (filled triangles). The data were fitted to a double exponential model, and the correlation constants for the fittings were 0.9994 (filled squares), 0.9953 (open circles), and 0.9950 (filled triangles), respectively. The data with error bars are expressed as the mean ± S.D. (n = 3–5).

crowding agents. The results indicate that single crowding agents remarkably decelerate the fast track of the refolding of MM-CK but accelerate the

To compare the refolding yield of MM-CK in mixed crowded solutions with that in single crowded solutions, we chose three mixed macromolecular crowding agents containing both CT DNA and polysaccharide (or PEG 2000). Denatured MM-CK was refolded in the presence of each of the mixed crowding agents. As shown in Figure 5(a), when the total concentration of the mixed crowding agent is 100 g/l, in which the weight ratio of CT DNA to Ficoll 70 is 1:9, the refolding yield of MM-CK after refolding for 3 h under this condition increased 23% compared with that in the presence of 10 g/l CT DNA, 18% compared with in the presence of 100 g/l Ficoll 70, and 19% compared with that in the absence of crowding agents. In the presence of 20 g/l CT DNA and 80 g/l Ficoll 70, the refolding yield of MM-CK after refolding for 3 h increased 18% compared with that in the presence of 20 g/l CT DNA, 6.5% compared with in the presence of 100 g/l Ficoll 70, and 6.9% compared with that in the absence of crowding agents. As can be seen from Figure 6(a), when the total concentration of the mixed crowding agent is 100 g/l, in which the weight ratio of CT DNA to dextran is 1:9, the refolding yield of MM-CK after refolding for 3 h under this condition increased 17% compared with that in the presence of 10 g/l CT DNA, 14% compared with in the presence of 100 g/l dextran 70, and 13% compared with that in the absence of crowding agents. As shown in Figure 7(a), in the presence of 10 g/l CT DNA and 90 g/l PEG 2000, the refolding yield of MMCK after refolding for 3 h under this condition increased 17% compared with that in the presence of 10 g/l CT DNA, 52% compared with in the presence of 100 g/l PEG 2000, and 13% compared with that in the absence of crowding agents. A remarkable increase in the refolding yield of

Table 1. Kinetic parameters of MM-CK refolding in the absence and in the presence of single crowding agents Crowding agent

Concentration (g/l)

Ymax,1 (%)

k1 (10−3 s−1)

(t1/2)1 (min)

Ymax,2 (%)

k2 (10−3 s−1)

(t1/2)2 (min)

Ymax,1 + Ymax,2 (%)

Absence dextran 70

0 100 200 100 200 100 200 10 20

60.6 ± 2.3 54.3 ± 0.7 38.4 ± 1.3 59.8 ± 1.0 53.0 ± 7.7 39.4 ± 3.3 25.9 ± 7.8 55.5 ± 1.8 53.8 ± 2.5

156 ± 8 14.9 ± 0.7 19.0 ± 1.5 13.8 ± 0.9 12.8 ± 3.0 11.9 ± 2.8 7.2 ± 5.5 18.0 ± 1.7 15.6 ± 2.6

4.45 ± 0.23 46.6 ± 2.0 36.5 ± 3.0 50.3 ± 3.1 54 ± 13 58.3 ± 13.7 96 ± 73 38.6 ± 3.6 44.3 ± 7.2

7.4 ± 1.9 18.4 ± 0.8 9.6 ± 1.7 12.7 ± 1.2 15.3 ± 9.7 17.0 ± 1.8 14.1 ± 3.0 12.0 ± 2.2 10.5 ± 3.5

21.9 ± 9.8 337 ± 37 179 ± 52 328 ± 80 50.2 ± 20 374 ± 87 198 ± 79 418 ± 194 230 ± 160

32 ± 14 2.1 ± 0.2 3.87 ± 1.13 2.11 ± 0.51 13.8 ± 5.5 1.9 ± 0.4 3.51 ± 1.40 1.66 ± 0.77 3.1 ± 2.2

68.0 ± 4.2 72.7 ± 1.5 48.0 ± 3.0 72.5 ± 2.2 68.3 ± 17.4 56.4 ± 5.0 40.0 ± 10.8 67.5 ± 4.0 64.3 ± 6.0

Ficoll 70 PEG 2000 CT DNA

Kinetic parameters were determined by fitting the increase in enzymatic activity versus time to a double exponential model as described in Materials and Methods. Data are expressed as mean ± S.D. (n = 3-5).

474

Mixed Crowding Accelerates MM-CK Refolding

four crowding agents were used in high concentrations, but it is not clear what combinations would be ideal to promote optimal refolding of the enzyme. In this study, both CT DNA and polysaccharide (or PEG 2000) were added to the refolding buffer, keeping the total concentration of the mixed crowding agent constant at 100 g/l, whereas the weight ratio of CT DNA to polysaccharide (or PEG 2000) varies gradually. As shown in Figure 8(a), (b), and (c), the refolding yield of denatured MM-CK after

Figure 5. Effect of mixed macromolecular crowding agents containing both CT DNA and Ficoll 70 on the reactivation of denatured MM-CK. (a) Yield of MM-CK refolding in the absence of crowding agents (–) and in the presence of 10 g/l CT DNA and 90 g/l Ficoll 70 (D1 + F), 20 g/l CT DNA and 80 g/l Ficoll 70 (D2 + F), 10 g/l CT DNA (D1), 20 g/l CT DNA (D2) or 100 g/l Ficoll 70 (F) after refolding for 3 h. (b) Refolding kinetics of MM-CK in the absence of crowding agents (filled squares) and in the presence of 10 g/l CT DNA and 90 g/l Ficoll 70 (open inverse triangles), 20 g/l CT DNA and 80 g/l Ficoll 70 (filled pentagrams), 10 g/l CT DNA (open circles), 20 g/l CT DNA (open triangles) or 100 g/l Ficoll 70 (filled rhombus). The data were fitted to a double exponential model, and the correlation constants for the fittings were 0.9994 (filled squares), 0.9951 (open inverse triangles), 0.9881 (filled pentagram), 0.9953 (open circles), 0.9950 (open triangles), and 0.9990 (filled rhombus), respectively. The inset shows the early kinetics of MM-CK in the presence of 10 g/l CT DNA and 90 g/l Ficoll 70 (open inverse triangles), 10 g/l CT DNA (open circles), or 100 g/l Ficoll 70 (filled rhombus) more clearly. The data with error bars are expressed as the mean ± S.D. (n = 3–5).

MM-CK after refolding for 3 h by a mixed crowding agent containing 20 g/l CT DNA and 80 g/l dextran 70 (or PEG 2000) was also observed (Figures 6(a) and 7(a)). Noticeable increases in the total maximal refolding yield of denatured MM-CK (Ymax,1 + Ymax,2) by these three mixed crowding agents were also observed (Table 2). Clearly there was a difference in the recovery of MM-CK activity when combinations of these

Figure 6. Effect of mixed macromolecular crowding agents containing both CT DNA and dextran 70 on the reactivation of denatured MM-CK. (a) Yield of MM-CK refolding in the absence of crowding agents (–) and in the presence of 10 g/l CT DNA and 90 g/l dextran 70 (D1 + d), 20 g/l CT DNA and 80 g/l dextran 70 (D2 + d), 10 g/l CT DNA (D1), 20 g/l CT DNA (D2) or 100 g/l dextran 70 (d) after refolding for 3 h. (b) Refolding kinetics of MM-CK in the absence of crowding agents (filled squares), in the presence of 10 g/l CT DNA and 90 g/l dextran 70 (open inverse triangles), 20 g/l CT DNA and 80 g/l dextran 70 (filled pentagram), 10 g/l CT DNA (open circles), 20 g/l CT DNA (filled triangles) or 100 g/l dextran 70 (filled rhombus). The data were fitted to a double exponential model, and the correlation constants for the fittings were 0.9994 (filled squares), 0.9979 (open inverse triangles), 0.9977 (filled pentagrams), 0.9953 (open circles), 0.9950 (open triangles), and 0.9995 (filled rhombus), respectively. The inset shows the early kinetics of MM-CK in the presence of 10 g/l CT DNA and 90 g/l dextran 70 (open inverse triangles), 10 g/l CT DNA (open circles), or 100 g/l dextran 70 (filled rhombus) more clearly. The data with error bars are expressed as the mean ± S.D. (n = 3–5).

475

Mixed Crowding Accelerates MM-CK Refolding

was ideal to promote optimal refolding of denatured MM-CK. Effects of mixed macromolecular crowding agents on refolding kinetics of MM-CK

Figure 7. Effect of mixed macromolecular crowding agents containing both CT DNA and PEG 2000 on the reactivation of denatured MM-CK. (a) Yield of MM-CK refolding in the absence of crowding agents (–) and in the presence of 10 g/l and 90 g/l PEG 2000 (D1 + P), 20 g/l CT DNA and 80 g/l PEG 2000 (D2 + P), 10 g/l CT DNA (D1), 20 g/l CT DNA (D2) or 100 g/l PEG 2000 (P) after refolding for 3 h. (b) Refolding kinetics of MM-CK in the absence of crowding agents (filled squares), in the presence of 10 g/l CT DNA and 90 g/l PEG 2000 (open inverse triangles), 20 g/l CT DNA and 80 g/l PEG 2000 (filled pentagrams), 10 g/l CT DNA (open circles), 20 g/l CT DNA (filled triangles) or 100 g/l PEG 2000 (filled rhombus). The data were fitted to a double exponential model, and the correlation constants for the fittings were 0.9994 (filled squares), 0.9979 (open inverse triangles), 0.9979 (filled pentagrams), 0.9953 (open circles), 0.9950 (open triangles), and 0.9972 (filled rhombus), respectively. The inset shows the early kinetics of MM-CK in the presence of 10 g/l CT DNA and 90 g/l PEG 2000 (open inverse triangles), 10 g/l CT DNA (open circles), or 100 g/l PEG 2000 (filled rhombus) more clearly. The data with error bars are expressed as the mean ± S.D. (n = 3–5).

refolding for 3 h changed non-linearly, increased from 67% to 79%/76%/75% when the weight ratio of CT DNA to the mixed crowding agent (CT DNA + Ficoll 70/dextran 70/PEG 2000) is between 0 and 10%, and then decreased gradually from 79%/ 76%/75% to 63%/62%/52% when the ratio is between 10% and 30%. Therefore, the combination of these crowding agents with a weight ratio of 1:9 (10 g/l CT DNA and 90 g/l polysaccharide or PEG)

As described above, denatured MM-CK refolded in the presence of each of the three mixed macromolecular crowding agents with a weight ratio of 1:9 (10 g/l CT DNA and 90 g/l polysaccharide or PEG) leads to a higher refolding yield. Therefore, we chose the weight ratio of 1:9 for the mixed crowding agents to compare the refolding kinetics of MM-CK in mixed crowded solutions with that in single crowded solutions. The refolding of MM-CK follows the biphasic first-order mechanism in the presence of such macromolecular crowding agents. The difference of refolding kinetics between these mixed and single crowding agents was clearly observed as shown in Figures 5(b), 6(b), and 7(b), from which the kinetic parameters of MM-CK refolding were determined. As can be seen from the inset of Figures 5(b), 6(b), and 7(b), these three mixed macromolecular crowding agents remarkably enhanced the initial rate for MM-CK refolding, compared with single crowding agents. Table 2 summarizes the kinetic parameters for the refolding of denatured MM-CK in the presence of such mixed crowding agents. As shown in Table 2, in the presence of a mixed crowding agent containing 10 g/l CT DNA and 90 g/l Ficoll 70, the rate for the fast track of MMCK refolding was increased 1.93 and 2.52-fold, and that for the slow track was increased 1.04 and 1.32fold, compared with those in the presence of 10 g/ l CT DNA and 100 g/l Ficoll 70, respectively. Furthermore, the rates for both tracks of MM-CK refolding in the presence of a mixed crowding agent containing 20 g/l CT DNA and 80 g/l Ficoll 70 were faster than those in the presence of single crowding agents. A remarkable increase in the rates for both tracks of MM-CK refolding by a mixed crowding agent containing 10 g/l CT DNA and 90 g/l dextran 70 was also observed (Table 2). The rate for the fast track of MM-CK refolding in the presence of a mixed crowding agent containing 10 g/l CT DNA and 90 g/ l PEG 2000 was slightly decreased and increased 1.15fold, and that for the slow track was increased 1.02 and 1.14-fold, compared with those in the presence of 10 g/l CT DNA and 100 g/l PEG 2000, respectively (Table 2). The results indicate that these three mixed crowding agents remarkably accelerated the refolding of MM-CK, compared with single crowding agents. Effect of mixed macromolecular crowding agents on aggregation during MM-CK refolding Crowding theory predicts that aggregation should increase under conditions of macromolecular crowding, since intramolecular excluded volume will increase the chemical potential of the unfolded state relative to that of the native or

476

Mixed Crowding Accelerates MM-CK Refolding

Table 2. Kinetic parameters of MM-CK refolding in presence of mixed or single crowding agents Mixed crowding agent concentration (g/l) CT DNA Ficoll 70 dextran 70 PEG 2000 10 20 0 10 20 0 10 20 0 10 20

0 0 100 90 80 0 0 0 0 0 0

0 0 0 0 0 100 90 80 0 0 0

0 0 0 0 0 0 0 0 100 90 80

Ymax,1 (%)

k1 (10−3 s−1)

(t1/2)1 (min)

Ymax,2 (%)

55.5 ± 1.8 53.8 ± 2.5 59.8 ± 1.0 59.3 ± 5.1 57.5 ± 3.2 54.3 ± 0.7 60.5 ± 1.5 61.4 ± 2.5 39.4 ± 3.3 67.9 ± 1.4 44.1 ± 2.5

18.0 ± 1.7 15.6 ± 2.6 13.8 ± 0.9 34.8 ± 4.9 18.8 ± 2.8 14.9 ± 0.7 20.0 ± 1.3 15.9 ± 1.9 11.9 ± 2.8 13.7 ± 1.1 14.9 ± 2.3

38.6 ± 3.6 44.3 ± 7.2 50.3 ± 3.1 20.0 ± 2.8 36.8 ± 5.4 46.6 ± 2.0 34.6 ± 2.3 43.7 ± 5.3 58.3 ± 13.7 50.7 ± 4.1 46.4 ± 7.0

12.0 ± 2.2 10.5 ± 3.5 12.7 ± 1.2 20.4 ± 5.4 17.3 ± 3.7 18.4 ± 0.8 16.5 ± 1.8 12.4 ± 3.4 17.0 ± 1.8 12.5 ± 1.7 25.3 ± 3.4

k2 (10−3 s−1) (t1/2)2 (min) 418 ± 194 230 ± 160 328 ± 80 434 ± 204 487 ± 270 337 ± 37 443 ± 116 202 ± 146 374 ± 87 427 ± 153 132 ± 27

1.7 ± 0.8 3.1 ± 2.2 2.1 ± 0.5 1.6 ± 0.7 1.4 ± 0.8 2.1 ± 0.2 1.6 ± 0.4 3.4 ± 2.5 1.9 ± 0.4 1.6 ± 0.6 5.2 ± 1.1

Ymax,1 + Ymax,2 (%) 67.5 ± 4.0 64.3 ± 6.0 72.5 ± 2.2 79.7 ± 3.5 74.8 ± 6.9 72.7 ± 1.5 76.9 ± 3.3 73.8 ± 5.9 56.4 ± 5.0 80.4 ± 3.1 69.3 ± 5.9

Kinetic parameters were determined by fitting the increase in enzymatic activity versus time to a double exponential model as described in Materials and Methods. Data are expressed as mean ± S.D. (n = 3–5).

compact non-native state (aggregates).30 Aggregation (turbidity) during the refolding of MM-CK in the presence of mixed or single crowding agents was measured by assay of the absorbance at 400 nm. As shown in Figure 9, aggregation of refolding samples of denatured MM-CK in the presence of either mixed or single crowding agents was more serious than that in the absence of crowding agents and almost no aggregation was observed in the absence of crowding agents. However, aggregation of MM-CK in the presence of any of the three mixed macromolecular crowding agents (10 g/l CT DNA and 90 g/l polysaccharide or PEG) is less serious than that in the presence of a single crowding agent of the same concentration. The results correlate well with the refolding yields of MM-CK in the presence of such mixed macromolecular crowding agents.

environments, consistent with the prediction that macromolecular crowding enhances protein aggregation. By using light scattering3,22 as another indicator of aggregation, we obtained qualitatively similar results (data not shown), further supporting the conclusion reached by turbidity that macromolecular crowding enhances MM-CK aggregation. It should be pointed out that we did not measure the amount of aggregated MM-CK here. Also, we did not determine whether the aggregates represent complexes between CT DNA and unfolded MMCK. Future work trying to figure out how to measure the amount of aggregation in these mixed crowded solutions should lead to a better understanding of how MM-CK aggregation is formed in physiological environments. Effects of single crowding agents on MM-CK refolding

Discussion Macromolecular crowding enhances MM-CK aggregation The cellular environment comprises a heterogeneous mixture of proteins, nucleic acids, ribosomes, and carbohydrates (polysaccharides), each of which is likely to affect the folding mechanism of different proteins in a distinctive fashion.40 The stabilizing effect of crowding on activity is largely restricted to macromolecules,41 and the effect of crowding on reaction rate is complex and depends crucially on the nature of the reaction and on the concentration of crowding agents.6 The results of Minton support the notion that macromolecular crowding enhances protein aggregation, at the expense of correct folding,42 which accords with the conjecture that the effects of crowding in media containing a substantial volume fraction of macromolecules are likely to greatly enhance the probability that a partially unfolded protein would aggregate,43 and consequently be lost from the pool of functional protein.44 The present study demonstrated that MM-CK was prone to form aggregates in crowded

The effects of single crowding agents on the refolding of GdnHCl-denatured MM-CK are diverse and depend on the nature of the crowding agents used. There are two aspects of the nature of single crowding agents. The first one is the relative sizes and shapes of the crowding agents used. The effect of volume occupancy on available volume induced by macromolecular crowding is sensitive to the relative sizes and shapes of the occupying molecules.2 Compared with dextran 70, Ficoll 70 behaves much more like a rigid sphere.45 Ficoll 70 is a highly branched copolymer of two short building blocks, sucrose (a disaccharide) and epichlorohydrin (a three-carbon cross-linker), making it less flexible and more compact than dextran 70 on a molecular weight basis. In contrast, dextran 70, a flexible, longchain poly (D-glucose) with sparse, short branches, is better modeled as a rod-like particle, while PEG 2000 is satisfactorily modeled as an effectively spherical particle.14 CT DNA is a threadlike molecule, and the diameter of the DNA double helix is only 2 nm. Although the relative size of high molecular mass PEG (PEG 2000) is much smaller than those of dextran 70, Ficoll 70, and CT DNA, its effect of steric repulsion on protein refolding is

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Mixed Crowding Accelerates MM-CK Refolding

order of magnitude lower than those of other crowding agents used. Mixed macromolecular crowding enhances native state stability and refolding rates of MM-CK MM-CK used here is a homo-dimeric protein. Many experiments concerning the refolding of MMCK have been carried out during the past two

Figure 8. Effects of mixed macromolecular crowding agents on refolding yield of MM-CK. Refolding yield of denatured MM-CK as a function of the ratio (%) of the weight of CT DNA to the total weight of mixed crowding agents: CT DNA + Ficoll 70 (a), CT DNA + dextran 70 (b), and CT DNA + PEG 2000 (c). Refolding yield was measured by the recovery of enzymatic activity after refolding for 3 h. The data with error bars are expressed as the mean ± S.D. (n = 2–3).

stronger than those of polysaccharide and nucleic acid crowding agents. In other words, the excluded volume effects of high molecular mass PEG are more effective than those of polysaccharide and nucleic acid crowding agents, which is demonstrated here. The second aspect is the viscosity of the single crowding agents used. At the same concentration, the viscosity of Ficoll 70 is higher than that of PEG 2000.46 Because of the character of CT DNA, its viscosity is far higher than those of Ficoll 70 and PEG 2000.47 The effects of 10 g/l CT DNA on refolding kinetics of MM-CK are similar to those of 100 g/l dextran 70 and Ficoll 70. The concentration of CT DNA as a crowding agent is one

Figure 9. Effects of mixed macromolecular crowding agents on aggregation of MM-CK during refolding. Aggregation of refolding samples of denatured MM-CK determined in the absence and in the presence of macromolecular crowding agents. (a) The agents used were 10 g/l CT DNA (D), 100 g/l PEG 2000 (P1), 90 g/l PEG 2000 (P2), and 10 g/l CT DNA + 90 g/l PEG 2000 (D + P2). (b) The agents used were 10 g/l CT DNA (D), 100 g/l dextran 70 (d1), 90 g/l dextran 70 (d2), and 10 g/l CT DNA + 90 g/l dextran 70 (D + d2). (c) The agents used were 10 g/l CT DNA (D), 100 g/l Ficoll 70 (F1), 90 g/l Ficoll 70 (F2), and 10 g/l CT DNA + 90 g/l Ficoll 70 (D + F2). N represents the aggregation in the absence of crowding agents. Aggregation (turbidity) of MM-CK was measured by the absorbance of the protein at 400 nm. The final enzyme concentration was 1.2 μM. The data with error bars are expressed as the mean ± S.D. (n = 2–3).

478 decades,38,39,48–51 but these experiments have all been carried out in dilute solutions. There is a wellestablished model for the refolding of MMCK.38,39,48 The first step of MM-CK refolding is very fast during which a burst phase intermediate, which is a partially folded monomer, is formed. Two rate-limiting steps follow this step: the dimerization of the two partially folded monomers and the formation of the active dimer with essentially the same conformation as the native state and with the activity recovered.39 The first rate-limiting step is the fast track of the refolding of MM-CK and the partially folded monomers are probably changed into misfolded dimers. The second rate-limiting step, the slow track of the refolding, is a very long and slow step to adjust the conformation of a dimeric partially folded intermediate to fold completely. The present study demonstrated that the presence of mixed crowding agents remarkably increased the refolding rates of both the fast track and the slow track of MM-CK refolding, compared with single crowding agents. Because the active dimer of MM-CK is more compact than the intermediate, the crowding theory predicts that macromolecular crowding enhances the stability of the native state relative to the intermediate and is favorable to the rearrangement of the protein. The refolding of MM-CK followed the biphasic firstorder mechanism in the presence of either mixed or single macromolecular crowding agents, indicating the existence of at least one intermediate during the refolding of MM-CK under crowding conditions. The crowding theory predicts that macromolecular crowding should enhance two aspects of protein folding: accelerating the initial collapse of polypeptide chains, whether newly synthesized inside the cell or refolding on dilution from denaturant in the test tube,3 and the association of partly folded chains into nonfunctional aggregates.6 The effect of excluded volume upon protein stability and conformation has been discussed using a simplified statistical-thermodynamic model.52 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 non-native state, resulting in enhancement of native or compact non-native state stability 30, 41 and refolding rates of globular proteins.19 But the biochemical experiments so far have demonstrated that the effects of macromolecular crowding on protein folding are very complex. For example, although lower refolding yields for the fast track of lysozyme refolding have been observed in the presence of high concentrations of mixed crowding agents, the refolding rates for the fast track are much faster than those in the absence of crowding agents.3 The present study demonstrated that mixed crowding agents containing CT DNA and polysaccharide (or PEG 2000) with suitable weight ratios accelerated the refolding process and increased the final refolding yield of denatured MM-CK in contrast to single crowding agents of the same

Mixed Crowding Accelerates MM-CK Refolding

concentration. These results suggest that the stabilization effects of mixed molecular crowding agents are stronger than those of single polysaccharide crowding agents. Ficoll 70 and dextran 70 have relative open structures,53 resulting in mixed crowding agents being less effective in excluding volume to the refolding molecules than single protein crowding agents.3 Mixed crowding reflects the physiological environments more accurately than individual crowding agents Here, the effects of Ficoll 70, dextran 70, PEG 2000, and CT DNA on the refolding of MM-CK were compared to one another, and to mixtures. Clearly there was an increase in the recovery of MM-CK activity when combinations of these crowding agents were used at high concentrations, but it is not clear how these agents work in combination. Here, an explanation why mixed crowding reflects the physiological environments more accurately than individual crowding agents is proposed. We suggest that these crowding agents work in concert to increase folding efficiency of MM-CK by increasing the available volume for the refolding proteins in a mixed crowded environment compared to that in a single crowded environment. It is evident that the available volume is a sensitive function of the relative sizes (and shapes) of test and background molecules.17,54 Tokuriki and colleagues have discussed in detail the effect of the chemical potential on the compaction of a test molecule in relation to the relative sizes of the test and background molecules, and have found that the maximum effect of crowding is observed if the size of the background molecule is similar to that of the test molecule.55 As such there are finite size effects on the magnitude of the crowding “pseudo-force” determined by the size of the crowding agent in relation to the size of the macromolecule of interest.3,56 The ability to dynamically change the size composition of a mixed crowded solution will hence posit an extra dimension for regulating the magnitude of the crowding effect and concomitantly for regulation of protein structure and enzyme function.3,56 Very recently, Li & Wu have used a density-functional theory (DFT) to investigate the structural and thermodynamic properties of concentrated electrolyte and neutral component mixtures that are highly asymmetric in terms of both size and charge mimicking a mixed crowded cellular environment.57 The DFT is able to take into account both the excluded volume effects and the longranged electrostatic interactions quantitatively. Although the DFT may not completely reflect many aspects of real systems, it represents at least a significant step forward towards understanding mixed macromolecular crowding. Because of its versatility to account for various intermolecular forces, the DFT approach appears promising for further theoretical studies of mixed macromolecular crowding under more realistic situations.

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Effect of possible complex formation between CT DNA and unfolded MM-CK on the refolding of MM-CK Because weak, non-specific interactions between macromolecular reactants and constituents of the local environment can greatly influence the equilibria and rates of reactions in which they participate,58 we investigated the effect of possible complex formation between CT DNA (which has a high negative charge density) and unfolded MM-CK on the refolding of MM-CK. The isoelectric point of MMCK is 6.9,59 thus the net charge of MM-CK is negative at pH 7.5 according to its amino acid composition. By using fluorescence spectroscopy41 we observed a weak, non-specific interaction between CT DNA and unfolded MM-CK at this pH, whose binding constant was 6.03(±0.06) × 10 3 , 5.78(±0.06) × 103, and 6.30(±0.07) × 103 M−1, respectively at 0, 0.15, and 1.0 M NaCl, approximately 6 orders of magnitude weaker than those of specific DNA–protein interactions.60,61 Since complex formation occurred, it is probably an important factor in the interpretation of refolding rates of MM-CK in the presence of nucleic acid crowding agents. The exact nature of this interaction is unknown, but the facts that MM-CK has a net negative charge and that unfolded MM-CK showed a weak and salt concentration-independent binding affinity to CT DNA make it unlikely to interact electrostatically with CT DNA at pH 7.5 used here. The concentration of CT DNA as a crowding agent is one order of magnitude lower than those of other crowding agents used, suggesting that, in addition to non-specific excluded volume effects, weak and non-specific DNA–protein interactions could greatly affect protein folding in the highly complex intracellular environment. Future work trying to elucidate the molecular mechanism of weak, nonspecific DNA–protein interactions affecting the refolding of MM-CK under mixed crowding conditions will help to provide answers to the interesting question “By how much does protein folding within cells differ from that in test tubes?”58 It remains unclear whether the accelerating effect we observed is just trivially due to a change in ionic strength accompanying the addition of a significant quantity of CT DNA, a strong electrolyte. The concentration of CT DNA we used was 24 mM or 48 mM, and polysaccharide (or PEG 2000) in the presence of 24 mM or 48 mM NaCl neither accelerated the refolding process nor increased the final refolding yield of denatured MM-CK in contrast to that in the absence of the salt (data not shown). Furthermore, a mixed macromolecular crowding agent (10 g/l CT DNA and 90 g/l Ficoll 70) in the presence of 50 mM or 150 mM NaCl neither accelerated the refolding process nor increased the final refolding yield of denatured MMCK in contrast to that in the absence of the salt (data not shown). Couthon et al. have also shown that MM-CK activity is unchanged by NaCl concentrations up to 1 M and is strongly inhibited when the

enzyme is incubated with NaCl concentrations higher than 3 M. 62 We thus suggest that the accelerating effect is not due to the change in ionic strength but results from mixed macromolecular crowding. Implications for protein folding in physiological environments First, the present study demonstrated that mixed crowding agents are less favorable to MM-CK aggregation than single crowding agents, consistent with the refolding yields of MM-CK under such mixed crowding conditions. Therefore, this paper provides evidence for the conclusion that mixed crowding reflects the physiological environments more accurately than individual crowding agents, more strongly than our previous study.3 Second, the present study suggested that, in addition to nonspecific excluded volume effects, non-specific nucleic acid–protein interactions could greatly affect protein folding in the highly complex intracellular environment, where large numbers of nucleic acids with very different properties are present. Therefore, this paper presents a new finding concerning the influence of background interactions58 upon the selected folding proteins within their native environments, as compared with our previous study.3 The manner in which a newly synthesized chain of amino acids transforms itself into a perfectly folded protein depends on both the intrinsic properties of the amino acid sequence and multiple contributing influences from the crowded cellular milieu. 63 Protein molecules, however, all have a finite tendency either to misfold or to fail to maintain their correctly folded states under some circumstances.64 Thus, correct folding and misfolding are competing processes during protein folding in intracellular environments. The findings that the refolding yield and rate of MM-CK increased remarkably and the aggregation extent decreased noticeably in a mixed macromolecular crowding environment might well be important in vivo, since the ultimate fate of a refolding protein inside the cell is very subtly dependent on the balance between a number of competing processes.65 The extension of the study of mixed macromolecular crowding 3 to MM-CK refolding in the presence of chaperones, which are more effective in assisting protein refolding under crowded conditions,22,29 should lead to a better understanding of how proteins fold in their intracellular environments.

Conclusions We have investigated the effect of mixed crowding agents on the refolding of MM-CK: refolding rates (both fast track and slow track refolding), yield, and amount of aggregation. Both the refolding yield and the refolding rate of MM-CK in mixtures of these agents are increased relative to the individual agents by themselves, and the mixed crowding agents lead

480 to a less serious aggregation than single crowding agents do, indicating that mixed macromolecular crowding agents are more favorable to MM-CK folding than single crowding agents and can be used to reflect the physiological environment more accurately than individual crowding agents. This conclusion further supports our concept “mixed macromolecular crowding”.3 Information obtained here can enhance our understanding of how these mixed crowding agents work in concert to increase folding efficiency.

Materials and Methods

Mixed Crowding Accelerates MM-CK Refolding thod.67 The reaction system contained 40 mM creatine, 4 mM ATP, 5 mM MgAc2, 0.01% (w/v) bromothymol blue and 50 mM Tris–HCl buffer (pH 7.5). The enzymatic activity was monitored by absorbance change at 617 nm and at 25 °C using a UV-2550 Probe spectrophotometer (Shimadzu, Kyoto, Japan). The specific activity of the enzyme under the native condition (pH 7.5) was 119(±8) units mg−1 (n = 3). All enzymatic activities were normalized against and expressed as a percentage of 1.2 μM solution of native MM-CK in refolding buffer. Control experiments were performed to ensure that the crowding agents used had no influence on the activity assay. Determination of kinetic constants for MM-CK refolding

Materials Rabbit MM-CK was prepared and purified as described.66 Purified MM-CK was homogenous on SDS– 48 PAGE. The A1% was used for 1cm value of 8.8 at 280 nm protein concentration measurements. Creatine was obtained from Shanghai Chemical Reagent Factory (Shanghai, China) with purity 99% and the disodium salt of ATP was a Boehringer Ingelheim Bioproducts product (Heidelberg, Germany) with purity >99%. The crowding agents, dextran 70, Ficoll 70, CT DNA, and PEG 2000, were purchased from Sigma (Sigma-Aldrich Co, St. Louis, MO). GdnHCl was obtained from Promega (Promega Corporation, Madison, WI). Dithiolthreitol (DTT) was a Biomol product (Biomol Co., Hamburg, Germany). All other chemicals used were made in China and of analytical grade. All reagent solutions were prepared in 50 mM Tris–HCl buffer (pH 7.5).

The time course of the refolding of MM-CK in the presence of mixed or single crowding agents was determined. Because the refolding of denatured MMCK follows the biphasic first-order mechanism,38,39,48 the kinetic parameters for the refolding were determined by fitting the increase in enzymatic activity versus time to a double exponential model,23,28,68 Y = Ymax,1(1-e−k1 t ) + Ymax,2(1-e−k2t), using OriginLab Origin 7.0 software. Here, Y is the refolding yield measured at the time t, Ymax,1 and Ymax,2 represent the maximal refolding yields for the fast track and the slow track of MM-CK refolding, respectively, and k1 and k2 are the rate constants for the fast track and the slow track, respectively. Refolding time constants, (t1/2)1 and (t1/2)2, are defined as the half-times for the fast track and the slow track of MM-CK refolding, respectively, where (t1/2)1 = 0.6931/k1 and (t1/2)2 = 0.6931/k2.

Preparation for CT DNA solution

Measurement of MM-CK aggregation

The CT DNA fragments were dissolved in 50 mM Tris– HCl buffer (pH 7.5), and then the solution was sonicated for 5 min by ultrasonic processor (Sonics, VC-130, Newtown, CT). Agar gel electrophoresis showed that the length of DNA is 200 base pairs mainly. Since DNA is a strong electrolyte, we adjusted the pH of CT DNA solution accordingly to maintain a constant pH (pH 7.5) by the addition of 50 mM Tris before use. The highly concentrated solution of CT DNA (higher than 40 g/l), which is very viscous and sticky, is extremely difficult to achieve and to mix rapidly.

Aggregation of MM-CK during the refolding of denatured MM-CK (1.2 μM) in the presence of mixed or single crowding agents was followed by monitoring the turbidity at 400 nm using a UV-2550 Probe spectrophotometer (Shimadzu, Kyoto, Japan) at 25 °C. In all experiments, blanks were subtracted in order to correct for absorbance of buffer components (including crowding agents).

MM-CK refolding

Acknowledgements

Samples of 60 μM dimeric MM-CK were incubated in 50 mM Tris–HCl buffer (pH 7.5) containing 3 M GdnHCl and 1 mM EDTA at 4 °C overnight. Refolding was initiated by a rapid 50-fold dilution of the concentrated denatured MM-CK solution into the refolding buffer, resulting in final concentrations of 60 mM GdnHCl and 1.2 μM MMCK in the refolding buffer. The refolding buffer consisted of 50 mM Tris–HCl (pH 7.5), 1 mM EDTA, 1 mM DTT, and different concentrations of crowding agents. All refolding experiments were carried out at 25 °C. MM-CK activity assay Refolding samples of denatured MM-CK were assayed at different time intervals using a pH-colorimetry me-

We thank Professor Allen P. Minton (National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health) for his helpful suggestions and Dr Peter McPhie (National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health) for his critical reading of the manuscript. This work was supported by National Natural Science Foundation of China grants 30370309 and 90408012, Project 863 grant 2004AA404260 from the Ministry of Science and Technology of China, and Program for New Century Excellent Talents in University grant NCET-04-0670 from the Ministry of Education of China.

Mixed Crowding Accelerates MM-CK Refolding

References 1. Minton, A. P. (1992). Confinement as a determinant of macromolecular structure and reactivity. Biophys. J. 63, 1090–1100. 2. Ellis, R. J. & Minton, A. P. (2003). Cell biology: join the crowd. Nature, 425, 27–28. 3. Zhou, B. R., Liang, Y., Du, F., Zhou, Z. & Chen, J. (2004). Mixed macromolecular crowding accelerates the oxidative refolding of reduced, denatured lysozyme: implications for protein folding in intracellular environments. J. Biol. Chem. 279, 55109–55116. 4. Minton, A. P. (1995). Macromolecular crowding: a foreword. Biophys. Chem. 57, 1–2. 5. Medalia, O., Weber, I., Frangakis, A. S., Nicastro, D., Gerisch, G. & Baumeister, W. (2002). Macromolecular architecture in eukaryotic cells visualized by cryoelectron tomography. Science, 298, 1209–1213. 6. Ellis, R. J. (2001). Macromolecular crowding: an important but neglected aspect of the intracellular environment. Curr. Opin. Struct. Biol. 11, 114–119. 7. Zimmerman, S. B. & Trach, S. O. (1991). Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli. J. Mol. Biol. 222, 599–620. 8. Swaminathan, R., Hoang, C. P. & Verkman, A. S. (1997). Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion. Biophys. J. 72, 1900–1907. 9. Fulton, A. B. (1982). How crowded is the cytoplasm? Cell, 30, 345–347. 10. Record, M. T., Jr, Courtenay, E. S., Cayley, S. & Guttman, H. J. (1998). Biophysical compensation mechanisms buffering E. coli protein-nucleic acid interactions against changing environments. Trends Biochem. Sci. 23, 190–194. 11. Hildebrandt, E. R. & Cozzarelli, N. R. (1995). Comparison of recombination in vitro and in E. coli cells: measure of the effective concentration of DNA in vivo. Cell, 81, 331–340. 12. Minton, A. P. (2000). Implications of macromolecular crowding for protein assembly. Curr. Opin. Struct. Biol. 10, 34–39. 13. Hall, D. & Minton, A. P. (2003). Macromolecular crowding: qualitative and semiquantitative successes, quantitative challenges. Biochim. Biophys. Acta, 1649, 127–139. 14. Zimmerman, S. B. & Minton, A. P. (1993). Macromolecular crowding: biochemical, biophysical, and physiological consequences. Annu. Rev. Biophys. Biomol. Struct. 22, 27–65. 15. Morgan, A. L. & Katzman, M. (2000). Subterminal viral DNA nucleotides as specific recognition signals for human immunodeficiency virus type 1 and visna virus integrases under magnesium-dependent conditions. J. Gen. Virol. 81, 839–849. 16. Minton, A. P., Colclasure, G. C. & Parker, J. C. (1992). Model for the role of macromolecular crowding in regulation of cellular volume. Proc. Natl Acad. Sci. USA, 89, 10504–10506. 17. Minton, A. P. (2001). The influence of macromolecular crowding and macromolecular confinement on biochemical reactions in physiological media. J. Biol. Chem. 276, 10577–10580. 18. Minton, A. P. (1997). Influence of excluded volume upon macromolecular structure and associations in “crowded” media. Curr. Opin. Biotechnol. 8, 65–69.

481 19. Cheung, M. S., Klimov, D. & Thirumalai, D. (2005). Molecular crowding enhances native state stability and refolding rates of globular proteins. Proc. Natl Acad. Sci. USA, 102, 4753–4758. 20. Minton, A. P. (2005). Models for excluded volume interaction between an unfolded protein and rigid macromolecular cosolutes: macromolecular crowding and protein stability revisited. Biophys. J. 88, 971–985. 21. Minton, A. P. & Wilf, J. (1981). Effect of macromolecular crowding upon the structure and function of an enzyme: glyceraldehyde-3-phosphate dehydrogenase. Biochemistry, 20, 4821–4826. 22. van den Berg, B., Ellis, R. J. & Dobson, C. M. (1999). Effects of macromolecular crowding on protein folding and aggregation. EMBO J. 18, 6927–6933. 23. van den Berg, B., Wain, R., Dobson, C. M. & Ellis, R. J. (2000). Macromolecular crowding perturbs protein refolding kinetics: implications for folding inside the cell. EMBO J. 19, 3870–3875. 24. Galán, A., Sot, B., Llorca, O., Carrascosa, J. L., Valpuesta, J. M. & Muga, A. (2001). Excluded volume effects on the refolding and assembly of an oligomeric protein. GroEL, a case study. J. Biol. Chem. 276, 957–964. 25. Rivas, G., Fernandez, J. A. & Minton, A. P. (2001). Direct observation of the enhancement of noncooperative protein self-assembly by macromolecular crowding: indefinite linear self-association of bacterial cell division protein FtsZ. Proc. Natl Acad. Sci. USA, 98, 3150–3155. 26. Hatters, D. M., Minton, A. P. & Howlett, G. J. (2002). Macromolecular crowding accelerates amyloid formation by human apolipoprotein C-II. J. Biol. Chem. 277, 7824–7830. 27. del Álamo, M., Rivas, G. & Mateu, M. G. (2005). Effect of macromolecular crowding agents on human immunodeficiency virus type 1 capsid protein assembly in vitro. J. Virol. 79, 14271–14281. 28. Li, J., Zhang, S. & Wang, C. C. (2001). Effects of macromolecular crowding on the refolding of glucose-6-phosphate dehydrogenase and protein disulfide isomerase. J. Biol. Chem. 276, 34396–34401. 29. Ren, G., Lin, Z., Tsou, C. L. & Wang, C. C. (2003). Effects of macromolecular crowding on the unfolding and the refolding of D-glyceraldehyde-3-phosophospate dehydrogenase. J. Protein Chem. 22, 431–439. 30. Sasahara, K., McPhie, P. & Minton, A. P. (2003). Effect of dextran on protein stability and conformation attributed to macromolecular crowding. J. Mol. Biol. 326, 1227–1237. 31. Mazon, H., Marcillat, O., Vial, C. & Clottes, E. (2002). Role of C-terminal sequences in the folding of muscle creatine kinase. Biochemistry, 41, 9646–9653. 32. Liang, Y., Du, F., Sanglier, S., Zhou, B. R., Xia, Y., Van Dorsselaer, A. et al. (2003). Unfolding of rabbit muscle creatine kinase induced by acid. A study using electrospray ionization mass spectrometry, isothermal titration calorimetry, and fluorescence spectroscopy. J. Biol. Chem. 278, 30098–30105. 33. Wallimann, T., Wyss, M., Brdiczka, D., Nicolay, K. & Eppenberger, H. M. (1992). Intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands: the “phosphocreatine circuit” for cellular energy homeostasis. Biochem. J. 281, 21–40. 34. Ellington, W. R. (2001). Evolution and physiological roles of phosphagen systems. Annu. Rev. Physiol. 63, 289–325.

482

Mixed Crowding Accelerates MM-CK Refolding

35. Wallimann, T. & Hemmer, W. (1994). Creatine kinase in non-muscle tissues and cells. Mol. Cell. Biochem. 133–134, 193–220. 36. Putney, S., Herlihy, W., Royal, N., Pang, H., Aposhian, H. V., Pickering, L. et al. (1984). Rabbit muscle creatine phosphokinase. cDNA cloning, primary structure and detection of human homologues. J. Biol. Chem. 259, 14317–14320. 37. Rao, J. K., Bujacz, G. & Wlodawer, A. (1998). Crystal structure of rabbit muscle creatine kinase. FEBS Letters, 439, 133–137. 38. Zhou, H. M. & Tsou, C. L. (1986). Comparison of activity and conformation changes during refolding of urea-denatured creatine kinase. Biochim. Biophys. Acta, 869, 69–74. 39. Li, S., Bai, J. H., Park, Y. D. & Zhou, H. M. (2001). Aggregation of creatine kinase during refolding and chaperonin-mediated folding of creatine kinase. Int. J. Biochem. Cell Biol. 33, 279–286. 40. Minton, A. P. (1995). Confinement as a determinant of macromolecular structure and reactivity. II. Effects of weakly attractive interactions between confined macrosolutes and confining structures. Biophys. J. 68, 1311–1322. 41. Zhou, Y. L., Liao, J. M., Chen, J. & Liang, Y. (2006). Macromolecular crowding enhances the binding of superoxide dismutase to xanthine oxidase: implications for protein-protein interactions in intracellular environments. Int. J. Biochem. Cell Biol. 38, 1986–1994. 42. Minton, A. P. (2000). Protein folding: thickening the broth. Curr. Biol. 10, R97–R99. 43. Ellis, R. J. & Minton, A. P. (2006). Protein aggregation in crowded environments. Biol. Chem. 387, 485–497. 44. Ellis, R. J. (1997). Molecular chaperones: avoiding the crowd. Curr. Biol. 7, R531–R533. 45. Luby-Phelps, K., Castle, P. E., Taylor, D. L. & Lanni, F. (1987). Hindered diffusion of inert tracer particles in the cytoplasm of mouse 3T3 cells. Proc. Natl Acad. Sci. USA, 84, 4910–4913. 46. Kozer, N. & Schreiber, G. (2004). Effect of crowding on protein-protein association rates: fundamental differences between low and high mass crowding agents. J. Mol. Biol. 336, 763–774. 47. Busch, N. A., Kim, T. & Bloomfield, V. A. (2000). Diffusion of proteins in DNA solutions. 2. Green fluorescent protein in crowded DNA solutions. Macromolecules, 33, 5932–5937. 48. Fan, Y. X., Zhou, J. M., Kihara, H. & Tsou, C. L. (1998). Unfolding and refolding of dimeric creatine kinase equilibriumandkineticstudies.ProteinSci.7,2631–2641. 49. Zhu, L., Fan, Y. X. & Zhou, J. M. (2001). Identification of equilibrium and kinetic intermediates involved in folding of urea-denatured creatine kinase. Biochim. Biophys. Acta, 1544, 320–332. 50. Zhu, L., Fan, Y. X., Perrett, S. & Zhou, J. M. (2001). Relationship between kinetic and equilibrium folding intermediates of creatine kinase. Biochem. Biophys. Res. Comm. 285, 857–862. 51. Leydier, C., Clottes, E., Couthon, F., Marcillat, O., Ebel, C. & Vial, C. (1998). Evidence for kinetic intermediate states during the refolding of GdnHCl-denatured MM-creatine kinase. Characterization of a trapped monomeric species. Biochemistry, 37, 17579–17589.

52. Minton, A. P. (2000). Effect of a concentrated “inert” macromolecular cosolute on the stability of a globular protein with respect to denaturation by heat and by chaotropes: a statistical-thermodynamic model. Biophys. J. 78, 101–109. 53. Wenner, J. R. & Bloomfield, V. A. (1999). Crowding effects on EcoRV kinetics and binding. Biophys. J. 77, 3234–3241. 54. Minton, A. P. (1998). Molecular crowding: analysis of effects of high concentrations of inert cosolutes on biochemical equilibria and rates in terms of volume exclusion. Methods Enzymol. 295, 127–149. 55. Tokuriki, N., Kinjo, M., Negi, S., Hoshino, M., Goto, Y., Urabe, I. & Yomo, T. (2004). Protein folding by the effects of macromolecular crowding. Protein Sci. 13, 125–133. 56. Hall, D. (2006). Protein self-association in the cell: a mechanism for fine tuning the level of macromolecular crowding? Eur. Biophys. J. 35, 276–280. 57. Li, Z.-D. & Wu, J.-Z. (2004). Density-functional theory for the structures and thermodynamic properties of highly asymmetric electrolyte and neutral component mixtures. Phys. Rev. E, 70, 031109-1-9. 58. Minton, A. P. (2006). How can biochemical reactions within cells differ from those in test tubes? J. Cell Sci. 119, 2863–2869. 59. White, M. Y., Cordwell, S. J., McCarron, H. C. K., Prasan, A. M., Craft, G., Hambly, B. D. & Jeremy, R. W. (2005). Proteomics of ischemia/reperfusion injury in rabbit myocardium reveals alterations to proteins of essential functional systems. Proteomics, 5, 1395–1410. 60. Terry, B. J., Jack, W. E., Rubin, R. A. & Modrich, P. (1983). Thermodynamic parameters governing interaction EcoRI endonuclease with specific and nonspecific DNA sequences. J. Biol. Chem. 258, 9820–9825. 61. Frankel, A. D., Ackers, G. K. & Smith, H. O. (1985). Measurement of DNA-protein equilibria using gel chromatography: application to the HinfI restriction endonuclease. Biochemistry, 24, 3049–3054. 62. Couthon, F., Clottes, E. & Vial, C. (1997). High salt concentrations induce dissociation of dimeric rabbit muscle creatine kinase. Physico-chemical characterization of the monomeric species. Biochim. Biophys. Acta, 1339, 277–288. 63. Dobson, C. M. (2003). Protein folding and misfolding. Nature, 426, 884–890. 64. Dobson, C. M. (2004). Principles of protein folding, misfolding and aggregation. Semin. Cell. Dev. Biol. 15, 3–16. 65. Dobson, C. M. & Karplus, M. (1999). The fundamentals of protein folding: bringing together theory and experiment. Curr. Opin. Struct. Biol. 9, 92–101. 66. Yao, Q. Z., Zhou, H. M., Hou, L. X. & Tsou, C. L. (1982). Conformational changes of creatine kinase during guanidine denaturation. Sci. Sin. 25B, 1186–1193. 67. Yao, Q. Z. & Tsou, C. L. (1981). The determination of activity of creatine kinase using pH-colorimetry. Prog. Biochim. Biophys. 3, 52–56. 68. van den Berg, B., Chung, E. W., Robinson, C. V., Mateo, P. L. & Dobson, C. M. (1999). The oxidative refolding of hen lysozyme and its catalysis by protein disulfide isomerase. EMBO J. 18, 4794–4803.

Edited by K. Kuwajima (Received 30 May 2006; received in revised form 28 August 2006; accepted 3 September 2006) Available online 12 September 2006