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ABSTRACT: Protein disulfide isomerase (PDI) functions as an isomerase to catalyze thiol:disulfide exchange, as a chaperone to assist protein folding, and as a subunit of prolyl-4-hydroxylase and microsomal triglyceride transfer protein. At a lower concentration of 0.2 microm, PDI facilitated the aggregation of unfolded rabbit muscle creatine kinase (CK) and exhibited anti-chaperone activity, which was shown to be mainly due to the hydrophobic interactions between PDI and CK and was independent of the cross-linking of disulfide bonds. At concentrations above 1 microm, PDI acted as a protector against aggregation but an inhibitor of reactivation during CK refolding. The inhibition effect of PDI on CK reactivation was further characterized as due to the formation of PDI-CK complexes through intermolecular disulfide bonds, a process involving Cys-36 and Cys-295 of PDI. Two disulfide-linked complexes containing both PDI and CK were obtained, and the large, soluble aggregates around 400 kDa were composed of 1 molecule of tetrameric PDI and 2 molecules of inactive intermediate dimeric CK, whereas the smaller one, around 200 kDa, was formed by 1 dimeric PDI and 1 dimeric CK. To our knowledge this is the first study revealing that PDI could switch its conformation from dimer to tetramer in its functions as a foldase. According to the observations in this research and our previous study of the folding pathways of CK, a working model was proposed for the molecular mechanism of CK refolding catalyzed by PDI.
Journal of Biological Chemistry 05/2005; 280(14):13470-6. · 4.77 Impact Factor
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ABSTRACT: Strong aggregation occurred in the refolding route of arginine kinase (AK) denatured with 3 mol GdnHCl/L (GdnHCl, guanidine hydrochloride). The activity recovery of GdnHCl-denatured AK was very low and dependent on the protein concentration in the process of refolding. For denatured AK at 1.2 micromol/L concentration, the recovered activity yield was about 45.2% of the native enzyme, whereas at 5.2 micromol/L the activity recovery yield was only 20% of native activity. The nonionic detergent Triton X-100 and Tween 20 (< or = 100 mmol/L concentration) not only effectively blocked the aggregation but also enabled the denatured AK to recover most of its native activity. The kinetics of aggregate solubilization showed that there was an induction phase dependent on the detergent, but there was no dependency when detergent was absent. The apparent activity recovery had a cooperative relation with detergents in the process of refolding, which suggested the existence of some interaction between the detergent and the refolding intermediate. On the basis of the study results, a scheme of refolding was proposed.
Biochemistry and Cell Biology 04/2005; 83(2):140-6. · 2.67 Impact Factor
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ABSTRACT: Protein stability plays an extremely important role not only in its biological function but also in medical science and protein engineering. Osmolytes provide a general method to protect proteins from the unfolding and aggregation induced by extreme environmental stress. In this study, the effect of glycerol on protection of the model enzyme creatine kinase (CK) against heat stress was investigated by a combination of spectroscopic method and thermodynamic analysis. Glycerol could prevent CK from thermal inactivation and aggregation in a concentration-dependent manner. The spectroscopic measurements suggested that the protective effect of glycerol was a result of enhancing the structural stability of native CK. A further thermodynamic analysis using the activated-complex theory suggested that the effect of glycerol on preventing CK against aggregation was consistent with those previously established mechanisms in reversible systems. The osmophobic effect of glycerol, which preferentially raised the free energy of the activated complex, shifted the equilibrium between the native state and the activated complex in favor of the native state. A comparison of the inactivation rate and the denaturation rate suggested that the protection of enzyme activity by glycerol should be attributed to the enhancement of the structural stability of the whole protein rather than the flexible active site.
Biophysical Journal 11/2004; 87(4):2247-54. · 3.65 Impact Factor
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ABSTRACT: The properties of rice-derived ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) in different concentrations of hydrogen peroxide (H2O2) solutions have been studied. The results indicate that at low H2O2 concentrations (0.2-10 mM), the properties of rubisco (e.g., carboxylase activities, structure, and susceptibility to heat denaturation) change slightly. However, at higher H2O2 concentrations (10-200 mM), rubisco undergoes an unfolding process, including the loss of secondary and tertiary structure, forming extended hydrophobic interface, and leading to cross-links between large subunits. High concentrations of H2O2 can also result in an increase in susceptibility of rubisco to heat denaturation. Further pre-treatments with or without reductive reagents to rubisco show that the disulfide bonds in rubisco help to protect the enzyme from damage by H2O2 as well as other reactive oxygen species.
Biochemistry (Moscow) 11/2004; 69(10):1136-42. · 1.06 Impact Factor
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ABSTRACT: The refolding process and the equilibrium intermediates of urea-denatured arginine kinase (AK) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) intrinsic fluorescence, far-UV circular dichroism (CD), size-exclusion chromatography (SEC), and enzymatic activity. In dilute denaturant, two equilibrium refolding intermediates (I and N') were discovered, and a refolding scheme of urea-denatured AK was proposed. During the refolding of urea-denatured AK, the fluorescence intensity increased remarkably, accompanied by a significant blue shift of the emission maximum and a pronounced increase in molar ellipticity of CD at 222 nm. The first folding intermediate (I) was inactive in urea solution ranging between 2.4 and 3.0 M. The second (N') existed between a 0.4- and 0.8-M urea solution, with slightly increased activity. Neither the blue shift emission maximum nor the molar ellipticity of CD at 222 nm showed significant changes in these two regions. The two intermediates were characterized by monitoring the ANS binding ability in various residual urea solutions, and two peaks of the emission intensity were observed in urea solutions of 0.6 and 2.8 M, respectively. The SEC results indicated that a distribution coefficient (K(D)) platform existed in urea solutions ranging between 2.4 and 3.0 M urea, suggesting that there was a similarly apparent protein profile and size in the urea solution region. The refolding kinetics showed that the urea-denatured AK was in two-phase refolding. Proline isomerization occurred in the unfolding process of AK, which blocked the slow phase of refolding. These results suggested that the refolding process of urea-denatured AK contained at the least two equilibrium refolding intermediates.
Protein Science 08/2004; 13(7):1892-901. · 2.80 Impact Factor
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ABSTRACT: The refolding of aminoacylase denatured in 6M guanidine hydrochloride (GdnHCl) has been studied by measuring enzyme activity, fluorescence emission spectra, ANS fluorescence spectra and far-UV circular dichroism spectra. The results showed that GdnHCl-denatured aminoacylase could be refolded and reactivated by dilution. A refolding intermediate was observed for low concentrations of GdnHCl (between 0.5 and 1.2M). This refolding intermediate was characterized by an increased fluorescence emission intensity, a blue-shifted emission maximum, and by increased binding of the fluorescence probe 8-anilino-1-naphthalenesulfonate (ANS). The secondary structure of the intermediate was similar to that of the native enzyme, and was therefore quite similar to the molten globule state often found in the protein folding pathway. Combined with the previous evidence of existence of an intermediate during unfolding process, we therefore proposed that the unfolding and refolding of aminoacylase might share the same pathway. A comparison of the Apo-enzyme and Holo-enzyme showed that there was little effect of the zinc ion on the refolding of the aminoacylase. Our study, the first successful report of the refolding of this metalloenzyme, also showed that lowering the concentration and the temperature of the enzyme improved the refolding rate of aminoacylase. The system therefore provides a useful model to study the refolding of proteins with prosthetic groups.
The International Journal of Biochemistry & Cell Biology 08/2004; 36(7):1332-40. · 4.63 Impact Factor
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ABSTRACT: The effect of oxidized dithiothreitol (DTT) on the conformation and function of arginine kinase from shrimp Feneropenaeus chinensis was investigated with the methods of intrinsic fluorescence, ANS fluorescence, size exclusion chromatography (SEC), sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), and activity assay. The excess molecular oxidized dithiothreitol could result in a loss of activity and conformational change of arginine kinase. The oxidized arginine kinase was characterized by monitoring the changes of fluorescence emission wavelength (excitation wavelength: 295 nm) and the intensity of 1-anilino-8-naphthalenesulfonate (ANS) binding (excitation wavelength: 380 nm) to the protein. The results of fluorescence spectra showed that the presence of oxidized DTT could result in a marked change in the enzyme tertiary structure. The conformational changes of native and oxidized arginine kinase are induced by the presence of the full set of transition state analog (TSA) components. The results of size exclusion chromatography and SDS-PAGE indicated that no disulfide bond was formed among the protein molecules in the oxidized-DTT solution.
Biochemistry and Cell Biology 07/2004; 82(3):361-7. · 2.67 Impact Factor
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ABSTRACT: It is believed that denatured-reduced lysozyme rapidly forms aggregates during refolding process, which is often worked around by operating at low protein concentrations or in the presence of aggregation inhibitors. However, we found that low concentration buffer alone could efficiently suppress aggregation. Based on this finding, stable equilibrium intermediate states of denatured-reduced lysozyme containing eight free SH groups were obtained in the absence of redox reagents in buffer of low concentrations alone at neutral or mildly alkaline pH. Transition in the secondary structure of the intermediate from native-like to beta-sheet was observed by circular dichroism (CD) as conditions were varied. Dynamic light scattering and ANS-binding studies showed that the self-association accompanied the conformational change and the structure rich in beta-sheet was the intermediate state for aggregation, which could form either amyloid protofibril or amorphous aggregates under different conditions as detected by Electron Microscopy. Combining the results obtained from activity analysis, RP-HPLC and CD, we show that the activity recovery was closely related to the conformation of the refolding intermediate, and buffer of very low concentration (e.g. 10mM) alone could efficiently promote correct refolding by maintaining the native-like secondary structure of the intermediate state. This study reveals reasons for lysozyme aggregation and puts new insights into protein and inclusion body refolding.
The International Journal of Biochemistry & Cell Biology 06/2004; 36(5):795-805. · 4.63 Impact Factor
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ABSTRACT: The reduction of 5,5'-dithiobis-(2-nitrobenzoic acid)-modified arginine kinase by dithiothreitol has been investigated using the kinetic theory of the substrate reaction during modification of enzyme activity. The results show that the modified arginine kinase can be fully reactivated by an excess concentration of dithiothreitol in a monophasic kinetic course. The presence of ATP or the transition-state analog markedly slows the apparent reactivation rate constant, while arginine shows no effect. The results of ultraviolet (UV) difference and intrinsic fluorescence spectra indicate that the substrate arginine-ADP-Mg2+ can induce conformational changes of the modified enzyme but adding NO3- cannot induce further changes that occur with the native enzyme. The reactive cysteines' location and role in the catalysis of arginine kinase are discussed. It is suggested that the cysteine may be located in the hinge region of the two domains of arginine kinase. The reactive cysteine of arginine kinase may play an important role not in the binding to the transition-state analog but in the conformational changes caused by the transition-state analog.
Biochemical and Biophysical Research Communications 05/2004; 317(2):539-44. · 2.48 Impact Factor
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ABSTRACT: Previous studies have predicted five disulfide bonds in Aspergillus niger phytase (phy A). To investigate the role of disulfide bonds, intrinsic fluorescence spectra, far-ultraviolet circular dichroism (CD) spectra, and an enzyme activity assay were used to compare the differences of catalytic activity and conformational stability of phytase during denaturation in urea in the presence and absence of dithiothreitol (DTT). In the presence of 2 mM DTT, the inactivation and unfolding were greatly enhanced at the same concentration of denaturant. The fluorescence emission maximum red shift and decreases of ellipticity at 222 nm were in accord with the changes of catalytic activity. The kinetics of the unfolding courses were a biphasic process consisting of two first-order reactions in the absence of DTT and a monophasic process of a first-order reaction in the presence of DTT. The results suggested that the loss of enzymatic activity was most likely because of a conformational change, and that disulfide bonds played an important role in three-dimensional structure and catalytic activity.
Biochemistry and Cell Biology 05/2004; 82(2):329-34. · 2.67 Impact Factor
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ABSTRACT: The thermostability of an enzyme that exhibits phytase and acid phosphatase activities was studied. Kinetics of inactivation and unfolding during thermal denaturation of the enzyme were compared. The loss of phytase activity on thermal denaturation is most suggestive of a reversible process. As for acid phosphatase activities, an interesting phenomenon was observed; there are two phases in thermal inactivation: when the temperature was between 45 and 50 degrees C, the thermal inactivation could be characterized as an irreversible inactivation which had some residual activity and when the temperature was above 55 degrees C, the thermal inactivation could be characterized as an irreversible process which had no residual activity. The microscopic rate constants for the free enzyme and substrate-enzyme complex were determined by Tsou's method [Adv. Enzymol. Relat. Areas Mol. Biol. 61 (1988) 381]. Fluorescence analyses indicate that when the enzyme was treated at temperatures below 60 degrees C for 60 min, the conformation of the enzyme had no detectable change; when the temperatures were above 60 degrees C, some fluorescence red-shift could be observed with a decrease in emission intensity. The inactivation rates (k(+0)) of free enzymes were faster than those of conformational changes during thermal denaturation at the same temperature. The rapid inactivation and slow conformational changes of phytase during thermal denaturation suggest that inactivation occurs before significant conformational changes of the enzyme, and the active site of this enzyme is situated in a relatively fragile region which makes the active site more flexible than the molecule as a whole.
The International Journal of Biochemistry & Cell Biology 04/2004; 36(3):447-59. · 4.63 Impact Factor
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ABSTRACT: Protein thermal aggregation plays a crucial role in protein science and engineering. Despite its biological importance, little is known about the mechanism and pathway(s) involved in the formation of aggregates. In this report, the sequential events occurring during thermal unfolding and aggregation process of hemoglobin were studied by two-dimensional infrared correlation spectroscopy. Analysis of the infrared spectra recorded at different temperatures suggested that hemoglobin denatured by a two-stage thermal transition. At the initial structural perturbation stage (30-44 degrees C), the fast red shift of the band from alpha-helix indicated that the native helical structures became more and more solvent-exposed as temperature increased. At the thermal unfolding stage (44-54 degrees C), the unfolding of solvent-exposed helical structures dominated the transition and was supposed to be responsible to the start of aggregation. At the thermal aggregation stage (54-70 degrees C), the transition was dominated by the formation of aggregates and the further unfolding of the buried structures. A close inspection of the sequential events occurring at different stages suggested that protein thermal aggregation involves distinct regions.
Biophysical Journal 04/2004; 86(3):1682-90. · 3.65 Impact Factor
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ABSTRACT: Aminoacylase is a dimeric enzyme containing one Zn(2+) ion per subunit. The arginine (Arg)-induced unfolding of Holo-aminoacylase and Apo-aminoacylase has been studied by measurement of enzyme activity, fluorescence emission spectra and 1-anilino-8-naphthalenesulfonate (ANS) fluorescence spectra. Besides being the most alkaline amino acid, the arginine molecule contains a positively charged guanidine group, similar to guanidine hydrochloride, and has been used in many refolding systems to suppress protein aggregation. Our results showed that arginine caused the inactivation and unfolding of aminoacylase, with no aggregation during denaturation. A comparison between the unfolding of aminoacylase in aqueous and HCl (pH 7.5) arginine solutions indicated that the guanidine group of arginine had protein-denaturing effects similar to those of guanidine hydrochloride, which might help us understand the mechanism by which arginine suppresses incorrect refolding. The results showed that arginine-denatured aminoacylase could be reactivated and refolded correctly, indicating that arginine is as good a denaturant as the guanidine or urea for study of protein unfolding and refolding. Both the intrinsic fluorescence and the ANS fluorescence spectra showed that the arginine-unfolded aminoacylase formed a molten globule state in the presence of KCl, suggesting that intermediates exist during aminoacylase refolding. The results for the Apo-aminoacylase followed were similar to those for the Holo-enzyme, suggesting that Holo- and Apo-aminoacylase might have a similar unfolding and refolding pathway.
The International Journal of Biochemistry & Cell Biology 03/2004; 36(2):296-306. · 4.63 Impact Factor
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ABSTRACT: The trichloroacetic acid (TCA)-induced unfolding of aminoacylase was investigated by measurement of aggregation, enzyme activity, intrinsic fluorescence, 8-anilino-1-naphthalene sulfonate (ANS) binding, circular dichroism, and native polyacrylamide gel electrophoresis. The results showed that TCA caused inactivation and unfolding of aminoacylase. Intrinsic fluorescence results demonstrated that the TCA-induced transition of aminoacylase was characterized by two distinct stages during which the fluorescence emission maxima first redshifted to 338 nm and then blueshifted to 332 nm, close to that of native protein. ANS binding measurements revealed that TCA-denatured aminoacylase had a large hydrophobic area for TCA concentration near 2 mM. Comparison of the relative changes in wavelength shift and in the ANS intensity suggested the formation of a stable molten globule state of aminoacylase with a slightly disrupted tertiary structure and more hydrophobic surface than the native protein. Far-UV circular dichroism results provided further support that TCA induced the formation of two partially folded intermediates each with an enhanced native-like secondary structure. The results collectively suggest that a TCA-induced molten globule state is formed and stabilized during unfolding of aminoacylase and that association of the molten globule state may account for precipitation of the protein when denatured by TCA.
Journal of Protein Chemistry 12/2003; 22(7-8):669-75.
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ABSTRACT: Aspartate is an osmolyte found in some marine invertebrates and cyclostome fish. The aspartate-induced unfolding of N-acylamino acid amido hydrolase (aminoacylase) has been studied by measuring enzyme activity, fluorescence emission spectra, 8-anilino-1-naphthalenesulfonate (ANS) fluorescence spectra and far-UV circular dichroism (CD) spectra. The results showed that aspartate caused the inactivation and unfolding of aminoacylase. Surprisingly, increasing concentration of aspartate showed the "acid-induced folding", which used to be seen only in strong acids or salts at much lower pH. Although aspartate has the pI of 2.77 that is the lowest among all the free amino acids, it is actually a weak acid. It is thus of great interest why it causes this phenomenon to happen. The relative change of intrinsic fluorescence and ANS binding spectra have shown that there existed a stable molten globule state of aminoacylase with slightly disrupted tertiary structure and more hydrophobic surface. The molten globule state indicates that intermediates existed during aminoacylase refolding process. Unlike the other acids, such as trichloroacetic acid, there is no precipitation observed as the aspartate concentrations increased. It suggests the aspartate anions have an osmotic effect for the molten globule formed during unfolding process. Binding of aspartate anion to the protonated protein, which minimizes the intramolecular repulsion, might explain the osmotic effect of this amino acid in the nature. The results also showed the Apo-aminoacylase followed similar rules as Holo-enzyme, which suggested the zinc ion may play more important roles on activity other than structure.
The International Journal of Biochemistry & Cell Biology 12/2003; 35(11):1558-72. · 4.63 Impact Factor
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ABSTRACT: The arginine (Arg)-induced unfolding and the salt-induced folding of creatine kinase (CK) have been studied by measuring enzyme activity, fluorescence emission spectra, native polyacrylamide gel electrophoresis and size exclusion chromatography (SEC). The results showed that Arg caused inactivation and unfolding of CK, but there was no aggregation during CK denaturation. The kinetics of CK unfolding followed a one-phase process. At higher concentrations of Arg (>160 mM), the CK dimers were fully dissociated, the alkali characteristic of Arg mainly led to the dissociation of dimers, but not denaturation effect of Arg's guanidine groups on CK. The inactivation of CK occurred before noticeable conformational changes of the whole molecules. KCl induced monomeric and dimeric molten globule-like states of CK denatured by Arg. These results suggest that as a protein denaturant, the effect of Arg on CK differed from that of guanidine and alkali, its denaturation for protein contains the double effects, which acts not only as guanidine hydrochloride but also as alkali. The active sites of CK have more flexibility than the whole enzyme conformation. Monomeric and dimeric molten globule-like states of CK were formed by the salt inducing in 160 and 500 mM Arg H(2)O solutions, respectively. The molten globule-like states indicate that monomeric and dimeric intermediates exist during CK folding. Furthermore, these results also proved the orderly folding model of CK.
Biochimica et Biophysica Acta 11/2003; 1652(1):7-16. · 4.66 Impact Factor
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ABSTRACT: Unfolding and aggregation are basic problems in protein science with serious biotechnological and medical implications. Probing the sequential events occurring during the unfolding and aggregation process and the relationship between unfolding and aggregation is of particular interest. In this study, two-dimensional infrared (2D IR) correlation spectroscopy was used to study the sequential events and starting temperature dependence of Myoglobin (Mb) thermal transitions. Though a two-state model could be obtained from traditional 1D IR spectra, subtle noncooperative conformational changes were observed at low temperatures. Formation of aggregation was observed at a temperature (50-58 degrees C) that protein was dominated by native structures and accompanied with unfolding of native helical structures when a traditional thermal denaturation condition was used. The time course NMR study of Mb incubated at 55 degrees C for 45 h confirmed that an irreversible aggregation process existed. Aggregation was also observed before fully unfolding of the Mb native structure when a relative high starting temperature was used. These findings demonstrated that 2D IR correlation spectroscopy is a powerful tool to study protein aggregation and the protein aggregation process observed depends on the different environmental conditions used.
Biophysical Journal 10/2003; 85(3):1959-67. · 3.65 Impact Factor
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ABSTRACT: Using the methods of far-ultraviolet circular dichroism (CD) spectra, fluorescence spectra, and enzyme activity assays, the inactivation and conformational changes of creatine kinase (CK) induced by 1,1,1,3,3,3-hexafluoro-2-propanol (hexafluoroisopropanol (HFIP)) of different concentrations were investigated. To avoid the aggregation of CK that occurs with high HFIP, concentrations of 0%-5% HFIP were used in this study. The CD spectra showed that HFIP concentrations above 2.5% strongly induced the formation of secondary structures of CK. No marked conformational changes were observed at low concentrations of HFIP (0%-2.5%). After incubation with 0.2% HFIP for 10 min, CK lost most of its activity. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity described previously by Tsou was applied to study the kinetics of CK inactivation during denaturation by HFIP. The inactivation rate constants for the free enzyme and the substrate-enzyme complex were determined by Tsou's method. The results suggested that low concentrations of HFIP had a high potential to induce helices of protein and that the active site of the enzyme was situated in a limited and flexible region of the enzyme molecule that was more susceptible to the denaturant than was the protein as a whole.
Biochemistry and Cell Biology 10/2003; 81(5):327-33. · 2.67 Impact Factor
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ABSTRACT: Calf intestinal alkaline phosphatase (CIP) was denatured in 3.0 M guanidine hydrochloride for 2 h at 25 degrees C, before being diluted 20-fold with 0.1 M, pH 8.0, Tris-HCl buffer solution containing various effector molecules such as Mg2+, Zn2+, and nucleotide phosphate. The reactivation courses of the enzyme were investigated by the level of activity recovery, the recovery rate constant, and the relative standard deviation of the data. In the presence of effectors, the courses under reducing and nonreducing conditions of disulfide bonds of protein were compared. It was concluded that for CIP, Mg2+ is a more efficient inducer of reconstitution of the active site and appears to play a specific role. In addition, the present study discusses the differences in the refolding effectors between bacterial and mammalian enzymes.
Journal of Protein Chemistry 08/2003; 22(5):417-22.
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ABSTRACT: The equilibrium unfolding of calf intestinal alkaline phosphatase in guanidinium chloride (GdmCl) solutions was studied by following the fluorescence and ultraviolet difference spectra. At low concentrations of GdmCl (< 1.6 M), the fluorescence intensity decreased with a slight red shift of the emission maximum from 332 nm to 344 nm. An unfolding intermediate state was observed at a broad concentration range of GdmCl as a denaturant (between 1.6 and 2.6 M). This intermediate was characterized by increased fluorescence emission intensity, ultraviolet difference absorption at 236 nm and 260 nm, as well as increased binding to the protein and red shift of the fluorescence probe 1-anilinonaphthalene-8-sulfonic acid.
Journal of Protein Chemistry 08/2003; 22(5):405-9.
