Molecular mechanism for osmolyte protection of creatine kinase against guanidine denaturation

Transcription

1 Eur. J. Biochem. 268, (2001) q FEBS 2001 Molecular mechanism for osmolyte protection of creatine kinase against guanidine denaturation Wen-Bin Ou 1, Yong-Doo Park 1 and Hai-Meng Zhou 1,2 1 Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China; 2 Protein Science Laboratory of the Ministry of Education, School of Life Science and Engineering, Tsinghua University, Beijing, China The effects of osmolytes, including dimethysulfoxide, sucrose, glycine and proline, on the unfolding and inactivation of guanidine-denatured creatine kinase were studied by observing the fluorescence emission spectra, the CD spectra and the inactivation of enzymatic activity. The results showed that low s of dimethysulfoxide (, 40%), glycine (, 1.5 M), proline (, 2.5 M) and sucrose (, 1.2 M) reduced the inactivation and unfolding rate constants of creatine kinase, increased the change in transition free energy of inactivation and unfolding (DDG u ) and stabilized its active conformation relative to the partially unfolded state with no osmolytes. In the presence of various osmolytes, the inactivation and unfolding dynamics of creatine kinase were related to the protein s. These osmolytes protected creatine kinase against guanidine denaturation in a -dependent manner. The ability of the osmolytes to protect creatine kinase against guanidine denaturation decreased in order from sucrose to glycine to proline. Dimethysulfoxide was considered separately. This study also suggests that osmolytes are not only energy substrates for metabolism and organic components in vivo, but also have an important physiological function for maintaining adequate rates of enzymatic catalysis and for stabilizing the protein secondary and tertiary conformations. Keywords: osmolytes; creatine kinase; inactivation; unfolding; transition free energy. Protein folding is the process by which the amino-acid sequence of a protein determines the three-dimensional conformation of the functional protein [1]. The elucidation of the molecular mechanism of protein folding from a disordered polypeptide chain to the specific native state remains one of the major challenges in biochemistry [2,3] and is referred to as the translation of the second half of the genetic code [4]. Chemical chaperones, which are various low molecular mass compounds, have been shown to stabilize proteins in their native state and to protect them against thermal denaturation and aggregation in vitro [5 7]. One class of such compounds, referred to as cellular osmolytes, is produced in cells in response to osmotic shock [8]. Relevant examples include various polyols, sugars, polysaccharides, organic solvents, and various amino acids and their derivatives. These compounds appear to enhance the ability of cells to respond or adapt to different metabolic insults, due in part to their stabilizing effect on the protein conformation [9]. There have been several reports about the function of osmolytes in the unfolding process of some proteins in vivo and in vitro [8 16]. Osmolytes are known to stabilize proteins against aggregation. Schein described Correspondence to H.-M. Zhou, Protein Science Laboratory of the Ministry of Education, School of Life Science and Engineering, Tsinghua University, Beijing , China. Fax: , Tel.: , zhm-dbs@mail.tsinghua.edu.cn Abbreviations: CK, creatine kinase; ANS, 1-anilino- 8-naphthalenesulfonate; GB, glycine betaine; GdmCl, guanidinium hydrochloride; glycine, betaine; TMAO, trimethylamine N-oxide. (Received 5 June 2001, revised 11 September 2001, accepted 24 September 2001) effects of osmolytes on proteins and on the solvent properties of water [17]. Many potential stabilizing co-solutes for proteins have been investigated [17] that mainly affect the solvent properties of water as related to protein polarity and protein diffusion. Osmolytes as solvent additives can affect protein affinity for the hydrophobic surfaces of enzymes, as well as protein stability and solubility. A common mechanism in plants, animals and microorganisms for protecting proteins involves synthesis and intracellular accumulation of certain small organic solutes known as organic osmolytes [18,19]. The ability of organic osmolytes to protect proteins against denaturation is believed to be both generic and independent of the evolutionary history of the protein [20,21]. Any mechanism offering generalized protection of proteins against denaturation is of fundamental importance to their folding, stability, and function and is of major practical interest in biotechnology, evolutionary biology and biochemistry [21]. The theory that osmolytes became associated with particular proteins through natural selection implies that particular physicochemical properties of the stabilizing organic osmolyte solutions were selected for their ability to protect macromolecules and other components of the organism [10,18]. Osmolytes are classified as compatible osmolytes including polyl and free amino acids and counteracting osmolytes such as trimethylamine N-oxide (TMAO) [21]. Compatible osmolytes protect proteins subjected to threatening conditions such as extreme temperature fluctuations, excessive dryness or high salt environments, while counteracting osmolytes protect cellular proteins against urea inactivation [18,19,21]. Compatible and counteracting osmolytes may have different mechanisms for protecting

2 5902 W.-B. Ou et al. (Eur. J. Biochem. 268) q FEBS 2001 proteins because the relevant environmental stresses vary [21]. Protein folding was investigated using creatine kinase [22,23] (CK, EC ) which is a key enzyme for cellular energy metabolism, catalyzing the reversible phosphoryl transfer from phosphocreatine to ADP [24,25]. The enzyme is composed of two identical subunits, each having a molecular mass of < 43 kda. Its structure was recently resolved at 2.35 Å resolution using X-ray diffraction [26]. Extensive investigations have been carried out to understand the folding mechanism of creatine kinase [27 31]. CK is completely unfolded in either 6 M urea or 3 M guanidine hydrochloride (GdmCl) after 1 h [30,32,33]. The unfolding of guanidine or urea-denatured CK has been well recorded [29 31,34]. However, the influence of osmolytes on the unfolding of guanidine-denatured CK has not been studied. Osmolytes maintain adequate catalytic rates of proteins, a high level of regulatory responsiveness and a precise balance between stability and flexibility of the structure (tertiary conformation, subunit assembly and multiprotein complexes) [10]. Therefore, we suggested that osmolytes might have more important physiological functions for maintaining life development and evolution. The purpose of this study was to develop an understanding of the unfolding kinetics and the mechanisms of osmolyte protection of proteins against guanidine denaturation at the molecular level. The results showed that these osmolytes reduced the CK inactivation rate, increased the change of the transition free energy, stabilized its conformation against guanidine denaturation compared to the partially unfolded state and decreased the CK unfolding rate in a -dependent manner. In addition, dimethysulfoxide and proline prevented exposure of hydrophobic regions of CK. The results suggest that osmolytes are not only energy substrates for metabolism and organic components in vivo, but also exert an important physiological function for maintaining adequate rates of enzymatic catalysis and for stabilizing the protein s secondary and tertiary conformations. MATERIALS AND METHODS Materials Rabbit muscle creatine kinase was purified as described previously [32]. Protein s were determined using the absorption coefficient, 1 ¼ 8.8 [32]. Purified CK was homogeneous as determined by SDS/PAGE. Ultrapure GdmCl, Tris, 1-anilino-8-naphthalenesulfonate (ANS), sucrose, glycine, proline and ATP were from Sigma. All other reagents were local products of analytical grade. CK assay in the presence of different osmolytes The CK activity was assayed using the ph-colorimetry method [32]. CK was added to a 30-mM Tris/HCl/1 mm EDTA buffer (ph 8.0) containing 0.8 M GdmCl and various amounts of dimethysulfoxide, sucrose, glycine or proline. The enzymatic activity was measured in aliquots taken at suitable time intervals. The enzymatic activity was monitored by absorption at 597 nm with a PerkinElmer Lambda Bio U/V spectrophotometer. The reactions all occurred at 25 8C. Analysis of CK unfolding with various osmolytes The influence of osmolytes including dimethysulfoxide, sucrose, glycine and proline on the unfolding of guanidinedenatured CK was followed by observing their fluorescence emission spectra. The CK was denatured by and 3 M GdmCl for 8 h in the same buffer containing 10% dimethysulfoxide, 0.6 M sucrose, 1.5 M glycine or 2.5 M proline. An excitation wavelength of 295 nm was used for inspecting the CK tryptophan and cysteine fluorescence intensity. Intrinsic fluorescence emission spectra were recorded. In addition, CK was added to the same unfolding system containing GdmCl and different osmolytes including 5 50% dimethysulfoxide, M sucrose, M glycine or M proline. The intrinsic fluorescence spectra of samples treated by various s of the osmolytes were measured over time. The intrinsic fluorescence emission spectra of all the samples were recorded after they were incubated for 8 h. The final CK was 1.2 mm. The reactions all occurred at 25 8C. The intrinsic fluorescence emission spectra were measured with a Hitachi 850 spectrofluorometer using 1-cm path-length cuvettes. The effect of sucrose on the CK secondary structure was followed by measuring the CD spectra. CK was added to the same unfolding buffer containing GdmCl and various amounts of sucrose at 25 8C. CD spectra were measured after 8 h. The final CK was 1.2 mm. CD spectra were recorded on a Jasco 725 spectrophotometer with a 2-mm path-length cell over a wavelength range of nm. CK binding of ANS CK was added to the same unfolding system as the intrinsic fluorescence analysis containing GdmCl and various amounts of dimethysulfoxide, sucrose, glycine or proline for 8 h. The final CK was 1.2 mm 10 ml of ANS (4 mm) was added to the samples and the ANS fluorescence emission spectra were recorded after 30 min. The final ANS was 40 mm. ANS fluorescence emission spectra were recorded from 410 to 600 nm. Samples were excited at 380 nm. The reactions all occurred at 25 8C. RESULTS Inactivation kinetics of CK in the presence of various osmolytes The CK enzymatic activity was measured over time after the CK was diluted in a 30-mM Tris/HCl/1 mm EDTA buffer (ph 8.0) containing 0.8 M GdmCl and 5 40% dimethysulfoxide, M sucrose, M glycine or M proline. Figure 1 shows the residual activity after 1.5 h. The semi-logarithmic and inactivation curves for the different dimethysulfoxide, sucrose, glycine or proline s are not shown. The inactivation rate constants (k u ) were calculated from semilogarithmic plots of the data. According to Tams et al. [16], DDG u ¼ RTln (k u,none /k u,osmolyte ). Table 1 shows the changes in transition free energy of inactivation (DDG u ) obtained in the presence of various osmolyte s relative to the case with no osmolytes. The results show that the native CK activity

3 q FEBS 2001 Osmolyte protection of creatine kinaseq1 (Eur. J. Biochem. 268) 5903 Fig. 1. Inactivation of 0.8 M GdmCl-denatured CK in the presence of dimethysulfoxide, sucrose, glycine or proline. CK was denatured in 0.8 M GdmCl at 25 8C. Inactivation was initiated by adding native CK to the unfolding buffer in the absence (A) and presence (B F) of various amounts of osmolytes. The enzymatic activity was measured after 1.5 h. The activity of the native enzyme at the same was taken as 100%. The dimethysulfoxide (white bars) s for B F were 5%, 10%, 20%, 30% and 40%; the sucrose (cross hatched bars) s were 0.3, 0.6, 0.9 and 1.2 M respectively; the glycine (gray bars) s were 0.25, 0.5, 1 and 1.5 M, respectively; and the proline (black bars) s were 0.5, 1, 1.5 and 2 M, respectively. The final CK was 2.5 mm. rapidly decreased in the absence of osmolytes and was almost zero after 30 min. With increasing s of dimethysulfoxide, sucrose, glycine or proline in the unfolding systems, the CK inactivation rate gradually slowed, but the changes in transition free energy of inactivation increased more than in the case with partially unfolded CK without osmolytes. Thus, these four osmolytes significantly stabilize the CK conformation. Furthermore, Fig. 2. Inactivation kinetics of 0.8 M GdmCl-denatured CK in the presence of 30% dimethysulfoxide, sucrose, glycine or proline. The conditions and procedures were the same as for Fig. 1 except for adding native CK into the buffer in the absence (X) and presence of 30% dimethysulfoxide (1), sucrose (O), glycine (V)or proline (B). The final CK was 1 mm. The inset shows a semilogarithmic plot of the kinetic course of inactivation of GdmCl-denatured CK in the absence and presence of 30% dimethysulfoxide, sucrose, glycine or proline. Points were obtained by subtracting the contribution of the slow phase. the CK inactivation rate and the change in transition free energy were closely related to the level of osmolyte. Inactivation kinetics for different s of CK in the presence of osmolytes The CK was diluted 57-fold, 114-fold and 171-fold in the same buffer as the activity assay, containing 0.8 M GdmCl and 30% dimethysulfoxide, sucrose, glycine or proline. The enzymatic activity was measured in aliquots Table 1. Effect of different osmolytes on the inactivation of CK at 25 8C. CK inactivation rate constants in an unfolding buffer containing various amounts of dimethylsulfoxide (DMSO), sucrose, glycine or proline. k, inactivation rate constant ( 10 3 s 21 ). DDG u (kj:mol 21 ), the change in transition free energy of unfolding in the presence of various osmolytes. Osmolyte (M) Sucrose Glycine Proline DMSO DMSO k DDG u k DDG u k DDG u (%) k DDG u

4 5904 W.-B. Ou et al. (Eur. J. Biochem. 268) q FEBS 2001 Fig. 3. Semilogarithmic plots for different s of CK inactivated by GdmCl denaturation with no osmolytes. The conditions and procedures were the same as for Fig. 1 except for the addition of native CK to the buffer with no osmolytes. The final CK s were 1 mm (X), 2 mm (O) and 3 mm (V), respectively. taken at appropriate time intervals. Figure 2 and Table 2 show the inactivation dynamics of 1 mm CK in the presence of osmolytes with the same molar s except for that of dimethysulfoxide. The changes in the transition-free energy of unfolding (DDG u ) were calculated using the method described above. The inactivation rate constants (k u ) and the values of DDG u indicate that the ability of the tested osmolytes to protect CK against guanidine denaturation was, in decreasing order, 30% dimethysulfoxide, sucrose, glycine and proline. Dimethylsulfoxide displayed the strongest protective ability among the various osmolytes. Figure 3 shows the inactivation data for CK in the absence of osmolytes. The CK inactivation included fast and slow phases. The enzymatic activity was < 95% inactivated during the fast phase. Protein s had almost no effect on the inactivation rate during the fast phase, but significantly affected the CK inactivation times in the fast phase. For different protein s, the various osmolytes had different abilities to protect CK against guanidine denaturation. Higher CK s in the presence of osmolytes tended to have lower inactivation rates than the unfolded state with no osmolytes (Table 3). Fig. 4. Intrinsic fluorescence emission spectra of GdmCl-unfolded CK in dimethysulfoxide, sucrose, glycine or proline. Native CK was added to the unfolding buffer containing GdmCl (A) or 3 M GdmCl (B). Traces as follows: (1), native CK alone; (2), partially unfolded CK alone; (3), with 10% dimethysulfoxide; (4), with 0.6 M sucrose; (5), with 1.5 M glycine; (6), with 2.5 M proline. The final CK was 1.2 mm. The excitation wavelength was 295 nm. Intrinsic fluorescence emission spectra of unfolded CK were measured after 8h. Table 2. Effect of different osmolytes on the inactivation of CK at 25 8C. CK (1 mm) inactivation rate constants (k ) in an unfolding buffer containing various osmolytes obtained from semilogarithmic plots (Fig. 2). DDG u (kj:mol 21 ): The change in transition free energy of unfolding in the presence of different osmolytes. DMSO, dimethylsulfoxide. Absence of osmolytes 30% DMSO Sucrose Glycine Proline k ( 10 3 s 21 ) DDG u

5 q FEBS 2001 Osmolyte protection of creatine kinaseq1 (Eur. J. Biochem. 268) 5905 Table 3. Effect of different osmolytes on the inactivation rate constants of various s of CK at 25 8C. Inactivation rate constants (k 10 3 s 21 ) CK Absence of osmolytes 30% DMSO Sucrose Glycine Proline 1 mm mm mm Unfolding of CK in the presence of different osmolytes CK was denatured by and 3 M GdmCl for 8 h in the same buffer containing 10% dimethysulfoxide, 0.6 M sucrose, 1.5 M glycine or 2.5 M proline. The intrinsic fluorescence spectra are shown in Fig. 4 (/3 M GdmCl). The results show that the CK unfolding was closely related to the GdmCl. According to Yao et al. [32], native CK was completely unfolded in 3 M GdmCl, with its maximum peak red shifted from 333 nm to 355 nm. Except for proline, the osmolytes did not prevent extensive CK unfolding. However, at GdmCl, proline, glycine and sucrose, but not dimethysulfoxide, prevented some conformational changes of the partially unfolded CK such that its peak position only red shifted from 333 nm to 347 nm. The results also showed that the various osmolyte s resulted in various effects on the CK unfolding process. The intrinsic fluorescence emission spectra of 5 50% dimethysulfoxide, M sucrose, M glycine or M proline treated samples were recorded after they were incubated with GdmCl for 8 h at 25 8C. Table 4 shows the effects of the different osmolyte s on the partially unfolded CK conformation. Increasing s of glycine or proline assisted in maintaining the CK conformation in the unfolding systems, prevented red shifting of the maximum peak and decreased the intrinsic fluorescence intensity relative to the unfolded state. The peak for the native CK was 333 nm. In the presence of different s of dimethysulfoxide, sucrose, glycine or proline, the maximum peak of the partially unfolded CK changed from 350 to 343, 345, 338 and 334 nm, respectively. However, the native state does not seem to be stabilized by dimethysulfoxide and sucrose as there was no increase in the amount of the native state relative to the partially unfolded state. Higher s of proline inhibited CK denaturation by completely Fig. 5. Circular dichroism spectra of GdmCl unfolded CK in the presence of different s of sucrose. The procedures were the same as for Fig. 4 except that the native CK was added to the unfolding buffer in the absence (2) and presence of 0.3 M sucrose (3), 0.6 M sucrose (4) and 1.2 M sucrose (5) for 8 h. CD spectra of partially unfolded CK were measured over a wavelength range of nm. Native CK and the buffer are labeled as (1) and (6), respectively. Fig. 6. ANS fluorescence emission spectra of partially unfolded CK in the presence of various amounts of dimethysulfoxide, sucrose, glycine, or proline. CK was added to the same unfolding system containing GdmCl and various amounts of osmolytes for 8 h. ANS (4 mm) was then added to each sample for 30 min. Final CK and ANS s were 1.2 mm AND 40 mm, respectively. The excitation wavelength was 380 nm. Fluorescence emission spectra of CK were measured over a wavelength range of nm. The bars in A, from left to right, are for native CK, partially unfolded CK and ANS. The dimethysulfoxide (white bars) s for bars B F were 5, 10, 20, 30 and 40%; the sucrose (cross hatched bars) s were 0.3, 0.6 and 1.2 M, respectively; the glycine (gray bars) s were 0.25, 0.5, 1, 1.5 and 2 M, respectively; and the proline (black bars) s were 0.25, 0.5, 1, 2 and 4 M, respectively.

6 5906 W.-B. Ou et al. (Eur. J. Biochem. 268) q FEBS 2001 Table 4. Effect of various osmolytes on the unfolding of CK at 25 8C. I (nm) and II, peak position and maximum intensity of the intrinsic fluorescence emission spectra of the refolded CK in the presence of various osmolytes. DMSO, dimethylsufloxide. Osmolyte (M) Sucrose Glycine Proline DMSO DMSO I II I II I II (%) I II Native-CK Native-CK maintaining its native conformation. In the control compound, N-acetyl-L-tryptophan, increasing dimethysulfoxide s boosted the intrinsic fluorescence intensity, but did not affect the peak position. Secondary structure analysis of unfolded CK in the presence of different s of sucrose The effect of sucrose on the CK secondary structure was studied by CD spectra analysis (the CD spectra with dimethysulfoxide, glycine and proline could not be measured because of their inherited effects on spectra). CK was added to the same unfolding buffer containing GdmCl and various s of sucrose. The CD spectra were measured after 8 h. Increasing sucrose s effectively protected the CK secondary structure against GdmCl impairment (Fig. 5). Unfolding kinetics of CK in the presence of distinct osmolytes During the CK unfolding process, CK was added to the same unfolding system containing GdmCl and 5 25% dimethysulfoxide, M sucrose, M glycine or M proline. The intrinsic fluorescence spectra were measured over time. DDG u was calculated from the semilogarithmic curves for the dimethysulfoxide, sucrose, glycine or proline treated samples (not shown) using the method described earlier. The CK unfolding rate constants and DDG u obtained from the semilogarithmic curves are listed in Table 5. With increased s of dimethysulfoxide, sucrose, glycine or proline in the unfolding systems, the CK unfolding rate constants slowed from s 21 to , , and s 21, respectively. The changes in transition free energy of unfolding increased relative to partially unfolded CK without osmolytes from 0 to 5.35, 5.80, 8.62 and 7.06 kj:mol 21, respectively. Thus, these four osmolytes stabilize the CK conformation compared to the partially unfolded state with no osmolytes. The results also demonstrated that the kinetics of unfolding for CK depend on the of dimethysulfoxide, sucrose, glycine or proline. ANS binding fluorescence ANS fluorescence intensity measurements for various amounts of dimethysulfoxide, sucrose, glycine or proline Table 5. Effect of various osmolytes on the unfolding kinetics of CK at 25 8C. CK unfolding rate constant in an unfolding buffer containing various amounts of dimethylsulfoxide (DMSO), sucrose, glycine or proline. k, Unfolding rate constants ( 10 3 s 21 ). DDG u (kj:mol 21 ), the change in transition free energy of unfolding in the presence of various osmolytes. Osmolyte (M) Sucrose Glycine Proline DMSO DMSO k DDG u k DDG u k DDG u (%) k DDG u

7 q FEBS 2001 Osmolyte protection of creatine kinaseq1 (Eur. J. Biochem. 268) 5907 added during the CK unfolding were used for evaluating the exposure of the hydrophobic groups. CK was added to the same unfolding system containing GdmCl and various amounts of dimethysulfoxide, sucrose, glycine or proline for 8 h. ANS (4 mm) was added to the samples and the ANS fluorescence emission spectra were recorded after 30 min. The final ANS was 40 mm. The data show that increasing s of dimethysulfoxide and proline suppressed the exposure of the CK hydrophobic groups (Fig. 6). However, sucrose and glycine produced opposite results. In the negative control, 2 M glycine, 5 M proline and 0.6 M sucrose slightly reduced the ANS fluorescence background intensity, but 50% dimethysulfoxide slightly increased the background intensity. DISCUSSION Protein-structure-disrupting solutes such as urea, guanidine, SDS, and arginine [19] interact strongly with proteins at polar peptide linkages or anionic sites. These solutes favor increased protein surface area, the unfolding of tertiary structure and the dissociation of multiple subunits to maximize these energetically favorable protein solute interactions [10]. The unfolding process exposes the hydrophobic portions of the protein. This information complements data obtained from conventional GdmCl- and urea-denaturation studies. An important question therefore is the role that significant hydrophobic interactions of the denatured protein with solutes play in determining protein folding pathways [35]. Both the unfolding and inactivation of CK in guanidine solutions have been found to be first order reactions [32,33]. In 3 M GdmCl, the inactivation rate constant was greater than that of unfolding. At lower guanidine s (, ), the inactivation rate constants were only slightly affected, while the unfolding rate constants decreased markedly. The multiphasic courses of inactivation at lower guanidine s suggest the presence of partly active intermediates during denaturation [32]. For the native CK containing 20 Tyr and eight Trp residues, some of the Trp residues are buried while others are exposed and some of the Tyr residues are in the ionized state. Guanidine below did not expose the buried Try residues or significantly affect the ionized Tyr residues. At 3 M guanidine, the Trp residues were exposed and the ionization state of the Tyr residues was also affected; the peptide chain was fully unfolded [33]. Between 0.5 M and GdmCl, most of the dimers dissociated and were completely inactive. Partial subunits unfolded accompanying dissociation. In 1.5 M GdmCl, dissociation of dimers was complete [36], and at low guanidine s (, ), an equilibrium of unfolded intermediates formed [22,23,36 38]. Inactivation occurred before noticeable conformational change during denaturation of CK. Therefore, the present study shows that in 0.8 M and GdmCl, various osmolytes mainly affected the first order reaction kinetics of CK inactivation and unfolding, which was related to the dissociation of the active dimer and limited subunit unfolding. The unfolding rate constant is the most important measure of protein stability [16]. In the presence of various osmolytes, CK inactivation and unfolding rate constants of CK decreased as compared to the unfolded CK without osmolytes (Figs 1 and 2; Tables 1, 2 and 5). In addition, with increased osmolyte s, the inactivation and unfolding rate constants of the first order CK reactions also decreased. As shown in Fig. 2, various types of osmolytes can protect CK. At the same molar s, the ability of three osmolytes to protect CK are, in decreasing order, sucrose, glycine and proline based on the inactivation rate constants (k u ) and the changes in transition free energy (DDG u ). Dimethysulfoxide is an organic solvent used to stabilize protein conformation in a manner simliar to glycerol. Although dimethysulfoxide can not be compared with other osmolytes, because its was measured in different units, it increased DDG u and reduced K u. Therefore, dimethysulfoxide does stabilize CK during guanidine denaturation. The CK inactivation kinetics in 0.8 M guanidine were found to be a combination of two first order reactions including fast and slow phases (Fig. 3). The enzyme lost considerable activity in the fast phase and then underwent a slower phase of inactivation. Protein s had almost no effect on the inactivation rate in the fast phase but the CK inactivation times in the fast phase were affected by protein s. For different protein s, CK in the presence of various osmolytes had different inactivation rates. The higher protein s tended to produce slower inactivation rates. However, in the presence of osmolytes besides sucrose, the CK inactivation rate was maintained to a certain extent for enzyme s above a certain level (. 2 mm). Therefore, the difference between the kinetics of inactivation reflects the ability of each osmolyte to maintain the enzyme in the active conformation. This paper also studied the effects of various osmolytes on the equilibrium of CK unfolding. Guanidine and osmolyte s are very important in CK unfolding. In 3 M GdmCl, various osmolytes (except proline) did not protect CK from fully unfolding. However, at lower guanidine s (), the same of osmolyte (except for dimethysulfoxide) protected CK from fully unfolding. It is likely that the dimethysulfoxide was too low to have any effect. The data in Table 4 clearly shows the effect of the dimethysulfoxide. With increased s of dimethysulfoxide, DDG u gradually increased compared to the partially inactivated state with no osmolytes. During the inactivation of CK by 0.8 M GdmCl, 10% dimethysulfoxide provided some protection (Fig. 1). However, the same s of dimethysulfoxide did not protect CK from conformational changes in GdmCl solution. The residual activity of CK was measured after CK was incubated with 10% dimethysulfoxide and 0.8 M GdmCl for 1.5 h. CK retained < 5% of the native CK activity. However, the intrinsic fluorescence emission spectra were measured after 8 h. It is possible that CK had completely lost its activity by this time. Therefore, the CK conformational change caused by guanidine is consistent with its inactivation in the presence of 10% dimethysulfoxide. The other osmolytes had effects similar to those of dimethysulfoxide. Figures 4, 5 and 6 and Table 4 show the ability of the four osmolytes to protect CK against guanidine denaturation. Denaturation of CK by GdmCl exposed many hydrophobic groups on the protein surface and partially destroyed the CK conformation. ANS fluorescence is a powerful means to inspect the exposure of hydrophobic surfaces. Figure 6 shows that during CK unfolding,

8 5908 W.-B. Ou et al. (Eur. J. Biochem. 268) q FEBS 2001 dimethysulfoxide and proline inhibit the exposure of hydrophobic areas and these effect were dependent. However, glycine and sucrose increased the hydrophobic groups on the CK surface. The ANS fluorescence results showed that dimethysulfoxide and proline stabilized the CK conformation. The effect of osmolytes on protein stability was dependent on high s of osmolytes [39]. Osmolytes exerted their effects through two different mechanisms: (a) the properties of water changed at high s of osmolytes; (b) osmolyte-specific binding to the protein was predominately at low s [16,40]. Here, the osmolytes protected CK against guanidine denaturation in a dependent manner. Dimethysulfoxide is a strong stabilizing agent that partly protects (50%) the helical domain of hen egg-white lysozyme and stabilizes partially folded conformations of proteins [41]. Recently, the effect of dimethysulfoxide on the inactivation of elastase at ph 11.5 was studied. The inactivation rate constants of elastase are and min 21 in the absence and presence of 20% dimethysulfoxide (v/v), respectively, which indicates that dimethysulfoxide stabilizes the enzyme in the active conformation [42]. In addition, dimethysulfoxide interferes with the formation of scrapie prion protein and acts to stabilize the a helical conformation of cellular prion protein [9]. More recently, dimethysulfoxide was found to augment superantigen and conventional antigen presentations by HLA-DM-deficient as well as HLA-DM-sufficient antigenpresenting cells. The enhancement is regarded as a subtle effect of dimethysulfoxide on the stability of several different proteins at the cell surface [13]. Our finding validates dimethysulfoxide protection of CK against guanidine denaturation mainly through changing the inactivation and unfolding kinetics of CK. The effects of dimethysulfoxide on CK inactivation and unfolding have several possible explanations. Dimethylpropiothetin is viewed as an excellent compatible osmolyte that accumulates in certain marine algae in response to rising ambient salinity [43]. It bears no net charge and has a structure-stabilizing carboxylate group and a dimethyl center [10]. The characteristics of dimethysulfoxide are similar to those of dimethylpropiothetin. It also contains a dimethyl group for stabilizing the CK conformation. Secondly, dimethysulfoxide alters the inactivation and unfolding dynamics of CK and stabilizes the unfolding intermediates of CK. Thirdly, the addition of dimethysulfoxide, strongly excluded from the ordered water that surrounds proteins, stabilizes the protein structures. Sucrose is a carbohydrate that plays a role in energy metabolism in vivo and is a very viscous osmolyte. Carbohydrate osmolytes are common in cyanobacteria, fungi, algae and vascular plants. Glycerol and trehalose are also found in some animals facing water stress [10]. The thermal stability of proteins increases in the presence of sugars [6,7]. Sucrose increased the of GdmCl required to unfold the phosphoglycerate kinase (PGK). The addition of denaturants change the structure of the ordered water; sucrose can counteract this change and stabilized the PGK conformation [44]. The peptide backbone of proteins plays a dominant role in protein stabilization by making use of naturally occurring osmolytes. Transfer free-energies were measured to evaluate the energy differences between the native and the unfolded states of ribonuclease A in water and in osmolyte solutions. The side chains were found to favor exposure to the osmolyte in comparison to exposure in water, and in this sense the side chains favor protein unfolding. However, in sucrose solutions, the highly unfavorable exposure of the polypeptide backbone on unfolding opposes and overrides the side chain preference for denaturation and results in stabilization of the protein [45]. Sucrose also effectively increases both the denaturation temperature and storage stability [46 48]. In addition, the degree of structural protection conferred by sucrose correlates with the extent of hydrogen bonding between the sugar and the protein [49]. The current study shows that sucrose markedly decreased the inactivation and unfolding rate constants of CK and increased DDG u. It effectively maintained the enzyme in the active conformation. Combining our results with previous findings, sucrose protection of CK against guanidine denaturation was partially due to stabilizing of the unfolding intermediates and changes in the unfolding kinetics of CK. On the other hand, sucrose possibly counteracted the water structural changes caused by guanidine, thus stabilizing the CK conformation. Glycine and proline are compatible osmolytes, which can be present in a cell at high s without disturbing the functional and structural properties of proteins. Among the free amino acids, glycine, proline and alanine commonly occur in different organisms [10]. These two free amino acids and several other amino acids were found to have no significant effect on the functions of several enzymatic reactions [18,50,51]. However, arginine and lysine sharply increase K m values of phosphoenolpyruvate and other phosphorylated ligands [50]. This effect may have resulted from the complexing of phosphorylated ligands by arginine and lysine [50,52]. N,N 0 -Dimethylglycine was effective in protecting the keratinocyte growth factor (KGF) against thermal denaturation [45]. The growth of a lysa mutant was restored by addition of glycine betaine (GB) in minimal medium. The growth rate increased proportionally with the augmentation of the intracellular GB. The addition of 1 mm proline had a similar effect to that observed in the presence of GB [53]. Our results show that glycine and proline are highly effective stabilizers, decrease the inactivation and unfolding rate constants, increase DDG u and protect the CK conformation. CK may possibly be protected against guanidine denaturation by using glycine and proline to influence the dynamics of CK unfolding, resulting in stabilization of the unfolding intermediates of CK. However, the viscosity effects and the changes of the water structure must also be considered. The isoelectric point of CK is near ph 7.0, thus CK molecules contain little anionic change in ph 8.0 environment. Glycine and proline contain two stabilizers, ammonium and acetate, which are typically incorporated into protein molecules in uncharged or anionic states [10]. Although proline and glycine are both nonpolar amino acids, they produced opposite ANS fluorescence results. Glycine has the simplest amino-acid structure. In a protein, the minimal steric hindrance of the glycine side chain allows much more structural flexibility than the other amino acids. But proline represents the opposite structural extreme. The secondary amino (imino) group is held in a rigid conformation that reduces the structural flexibility of the protein at that point. In addition, glycine does not have a rotation due to the lack of an a carbon atom with chirality. Proline is a loop structure

9 q FEBS 2001 Osmolyte protection of creatine kinaseq1 (Eur. J. Biochem. 268) 5909 containing a secondary amino group and is capable of rotation. It is often found in the C-terminus of an a helix. Higher s of proline (. 1.5 M) can form loose, higher-order molecular aggregates. The supramolecular assembly of proline was found to possess an amphipathic character [54]. These chemical characteristics may provide an explanation for the distinct ANS fluorescence results using glycine and proline. The mechanism for TMAO protection of ribonuclease T1 against urea denaturation was studied by measuring the transfer free-energy [21]. TMAO is a solute concentrated in the urea-rich cells of elasmobranches and coelacanth. The favorable interaction of urea with the backbone provides the dominant driving force for protein unfolding by this denaturant, and the unfavorable interaction of TMAO with the backbone is the dominant force opposing urea denaturation. At higher TMAO s, the role of the peptide backbone was more important than that of side chains in counteracting urea denaturation and in stabilizing ribonuclease T1. Furthermore, the transfer free energy of the denatured ribonuclease A amino acids was measured in the water with and without osmolytes [14]. The side chains were found to favor exposure to the osmolytes over exposure to water, but the peptide backbone exhibited unfavorable transfer free-energy in the presence of osmolytes. The highly unfavorable exposure of the polypeptide backbone on unfolding counteracted the side chain effects and resulted in stabilization of the proteins and offset protein denaturation. The four osmolytes themselves stabilized protein elements, such as functional groups, molecular characteristics, chemical chaperone activity, CK inactivation, unfolding kinetics and changes in the transition free-energy of CK unfolding relative to the unfolded state with no osmolytes. In addition, highly unfavorable osmolyte peptide backbone interactions possibly effectively offset favorable interaction of the osmolytes with the amino-acid side chains that promoted protein denaturation because of the addition of guanidine. Thus, osmolytes not only opposed denaturation but also provided stabilization against denaturation by guanidine. Furthermore, the increasing osmolyte s in the unfolding systems caused a large change in the CK unfolding environments, mainly the viscosity and ionic strength. The competitive process among CK, the osmolytes and GdmCl indicated that protein solvent interactions also played a key role in the osmolyte protection of CK against guanidine denaturation. Commonly occurring organic osmolytes are strongly excluded from the ordered water that surrounds proteins and therefore stabilize the protein structures [10,40,55,56]. Interactions between the protein surface charges and the solvent molecules lead to a redistribution of the solvent molecules, as the protein charges were fixed by the packing of the tertiary structure [56]. Preferential interactions between proteins and mixed solvent systems lead to either hydration or solvation of the macromolecule [39,57]. Organic solvents whose dielectric constant and polarizability differ from those of bulk water modify both the water hydrogen bonding character and the dissociation constant of ionizable side chains [42]. The gliding movement of the hydration-water molecules along the protein surface is associated with the breakage and formation of hydrogen bonds [58]. Thus, the presence of osmolytes greatly modifies the side-chain hydrogen bonding potential and the polarization of the backbone permanent dipole, which induce an increase in the hydrogen bonding character of neighboring polar residues [59]. The consequences are local rearrangements of water molecules and stabilization of the oriented dipole, which both contribute to the stability of the whole molecule [42]. As a consequence, CK was stabilized in an unfolding buffer containing various amounts of osmolytes. These results and previous studies were used to suggest a molecular mechanism for osmolytes protection of CK against guanidine denaturation at low GdmCl s shown in Scheme 1: Scheme 1. U 2 is a partially inactive dimeric unfolding intermediate, U 0 is a fully inactive dimeric unfolding intermediate and U is an extensively unfolded CK subunit. Low s of guanidine induced the formation of the unfolding intermediates [23,37]. In addition, at low s of guanidine, the CK inactivation included fast and slow phases. The osmolytes mainly affected the first order reaction kinetics of CK inactivation and unfolding, altered the inactivation and unfolding dynamics of CK and stabilized the partially inactive CK unfolding intermediate, thereby maintaining an active CK conformation and protecting CK against guanidine denaturation. The results showed that osmolytes such as dimethysulfoxide, sucrose, glycine and proline decreased the CK inactivation rate, increased DDG u, prevented structural changes in the unfolding systems, and stabilized the CK conformations and dimeric states. The results suggest that sucrose, glycerol and the free amino acids are not only energy substrates and organic components in vivo, but also exert an important physiological function for maintaining adequate rates of enzymatic catalysis and for stabilizing the protein secondary and tertiary conformations. 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