Effect of osmolytes as folding aids on creatine kinase refolding pathway.

The influence of osmolytes, including dimethysulfoxide, glycine, proline and sucrose, on the refolding and reactivation courses of guanidine-denatured creatine kinase was studied by fluorescence emission spectra, circular dichroism spectra, recovery of enzymatic activity and aggregation. The results showed that low concentrations of dimethysulfoxide (<20%), glycine (<0.5 M), proline (<1 M) and sucrose (<0.75 M) improved the refolding yields of creatine kinase, but high osmolyte concentrations decreased its recovery. Sucrose favored the secondary structural formation of creatine kinase. Proline and sucrose facilitated refolding of the protein to its original conformation, while dimethysulfoxide and proline accelerated the hydrophobic collapse of creatine kinase to a packed protein. During the aggregation of creatine kinase, dimethysulfoxide and sucrose inhibited aggregation of creatine kinase, as did proline, but glycine was unable to inhibit aggregation. These systematic observations further support the suggestion that osmolytes, including low concentrations of dimethysulfoxide, proline or sucrose, possibly play a chaperone role in the refolding of creatine kinase. The results also indicate that sucrose and free amino acids are not only energy substrates and organic components in vivo, but also help correct protein folding.     

Role of osmolytes as chemical chaperones during the refolding of aminoacylase.

The refolding and reactivation of aminoacylase is particularly difficult because of serious off-pathway aggregation. The effects of 4 osmolytes--dimethylsulphoxide, glycerol, proline, and sucrose--on the refolding and reactivation of guanidine-denatured aminoacylase were studied by measuring aggregation, enzyme activity, intrinsic fluorescence spectra, 1-anilino-8-naphthalenesulfonate (ANS) fluorescence spectra, and circular dichroism (CD) spectra. The results show that all the osmolytes not only inhibit aggregation but also recover the activity of aminoacylase during refolding in a concentration-dependent manner. In particularly, a 40% glycerol concentration and a 1.5 mol/L sucrose concentration almost completely suppressed the aminoacylase aggregation. The enzyme activity measurements revealed that the influence of glycerol is more significant than that of any other osmolyte. The intrinsic fluorescence results showed that glycerol, proline, and sucrose stabilized the aminoacylase conformation effectively, with glycerol being the most effective. All 4 kinds of osmolytes reduced the exposure of the hydrophobic surface, indicating that osmolytes facilitate the formation of protein hydrophobic collapse. The CD results indicate that glycerol and sucrose facilitate the return of aminoacylase to its native secondary structure. The results of this study suggest that the ability of the various osmolytes to facilitate the refolding and renaturation of aminoacylase is not the same. A survey of the results in the literature, as well as those presented here, suggests that although the protective effect of osmolytes on protein activity and structure is equal for different osmolytes, the ability of osmolytes to facilitate the refolding of various proteins differs from case to case. In all cases, glycerol was found to be the best stabilizer and a folding aid.     

Efficient refolding of aggregation-prone citrate synthase by polyol osmolytes: how well are protein folding and stability aspects coupled?

Efficient refolding of proteins and prevention of their aggregation during folding are of vital importance in recombinant protein production and in finding cures for several diseases. We have used citrate synthase (CS) as a model to understand the mechanism of aggregation during refolding and its prevention using several known structure-stabilizing cosolvent additives of the polyol series. Interestingly, no parallel correlation between the folding effect and the general stabilizing effect exerted by polyols was observed. Although increasing concentrations of polyols increased protein stability in general, the refolding yields for CS decreased at higher polyol concentrations, with erythritol reducing the folding yields at all concentrations tested. Among the various polyols used, glycerol was the most effective in enhancing the CS refolding yield, and a complete recovery of enzymatic activity was obtained at 7 m glycerol and 10 mug/ml protein, a result superior to the action of the molecular chaperones GroEL and GroES in vitro. A good correlation between the refolding yields and the suppression of protein aggregation by glycerol was observed, with no aggregation detected at 7 m. The polyols prevented the aggregation of CS depending on the number of hydroxyl groups in them. Stopped-flow fluorescence kinetics experiments suggested that polyols, including glycerol, act very early in the refolding process, as no fast and slow phases were detectable. The results conclusively demonstrate that both the thermodynamic and kinetic aspects are critical in the folding process and that all structure-stabilizing molecules need not always help in productive folding to the native state. These findings are important for the rational design of small molecules for efficient refolding of various aggregation-prone proteins of commercial and medical relevance.     

Intermediate studies on refolding of arginine kinase denatured by guanidine hydrochloride.

The refolding course and intermediate of guanidine hydrochloride (GuHCl)-denatured arginine kinase (AK) were studied in terms of enzymatic activity, intrinsic fluorescence, 1-anilino-8-naphthalenesulfonte (ANS) fluorescence, and far-UV circular dichroism (CD). During AK refolding, the fluorescence intensity increased with a significantly blue shift of the emission maximum. The molar ellipticity of CD increased to close to that of native AK, as compared with the fully unfolded AK. In the AK refolding process, 2 refolding intermediates were observed at the concentration ranges of 0.8-1.0 mol/L and 0.3-0.5 mol GuHCl/L. The peak position of the fluorescence emission and the secondary structure of these conformation states remained roughly unchanged. The tryptophan fluorescence intensity increased a little. However, the ANS fluorescence intensity significantly increased, as compared with both the native and the fully unfolded states. The first refolding intermediate at the range of 0.8-1.0 mol GuHCl/L concentration represented a typical "pre-molten globule state structure" with inactivity. The second one, at the range of 0.3-0.5 mol GuHCl/L concentration, shared many structural characteristics of native AK, including its secondary and tertiary structure, and regained its catalytic function, although its activity was lower than that of native AK. The present results suggest that during the refolding of GuHCl-denatured AK there are at least 2 refolding intermediates; as well, the results provide direct evidence for the hierarchical mechanism of protein folding.     

Towards creatine kinase aggregation due to the cysteine modification at the flexible active site and refolding pathway

The dimeric native state of creatine kinase (CK) was aggregated at conspicuous levels during cysteine modification at the active site with using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) under a high enzyme concentration. Measuring the ANS-binding fluorescence revealed that the hydrophobic surface of CK was increased by cysteine modification due to the flexible active site, and this resulted in insoluble aggregation, probably via non-specific hydrophobic interactions. To determine whether the aggregates can be refolded, 3M guanidine hydrochloride (GdnHCl) was used to dissolve the aggregates into the denatured form. Refolding of the solubilized enzyme sample was then conducted, accompanied by deprivation of DTNB from the CK in the presence of DTT. As a result, CK was reactivated by up to 40% with partial recovery of the tertiary (78%) and secondary structures (77%). To further elucidate its kinetic refolding pathway, both time interval measurements and a continuous substrate reaction were performed. The results showed that the refolding behavior was similar to the manner of normal CK folding with respect to the following two-phase kinetic courses. Additionally, the rate constants for the dimerization of the unfolded CK were dependent on the enzyme concentration and this was irrespective to the DTT concentrations, suggesting the rate-limiting steps of CK reassociation. The present study will expand our insight into the flexibility of the enzyme active site, which might act as a risk factor for inducing the unfavorable aggregation and partial refolding pathway of CK, as well as inducing an intermediate-like state recovery from aggregation.     

Effect of Mg2+ during reactivation and refolding of guanidine hydrochloride-denatured creatine kinase

Creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) was completely denatured using 3 M guanidine hydrochloride for 2 h as in previous studies [Yao et al. (1982), Sci. Sin. 25B, 1296–1302; Yao et al. (1984), Biochemistry 23, 2740–2744; Yao et al. (1982), Sci. Sin. 25B, 1186–1193]. Under suitable conditions, about 60–70% of the activity can be recovered in the presence of different Mg²⁺ concentrations. Both the reactivation and the refolding processes follow two-phase courses after dilution in the proper solutions. A comparison of the rate constants for the refolding of unfolded creatine kinase with those for the recovery of its catalytic activity at various Mg²⁺ concentrations shows that these are not synchronized. The reactivity of guanidine hydrochloride-denatured creatine kinase can be inhibited by Mg²⁺; however, the rates of reactivation are independent of the Mg²⁺ concentration. In addition, Mg²⁺ affects the fluorescence intensity, but the rate constants of refolding are independent of Mg²⁺ concentration. Although the reactivation of GdHCl-denatured creatine kinase is complete about 3 h after dilution with reactivation solutions, the conformational changes during refolding occur in a much slower reaction. Mg²⁺ can induce complex changes in the relative fluorescence intensity during refolding over a broad range of concentrations.     

Chaperone-like activity of peptidyl-prolyl cis-trans isomerase during creatine kinase refolding

Porcine kidney 18 kD peptidyl-prolyl cis-trans isomerase (PPIase) belongs to the cyclophilin family that is inhibited by the immunosuppressive drug cyclosporin A. The chaperone activity of PPIase was studied using inactive, active, and alkylated PPIase during rabbit muscle creatine kinase (CK) refolding. The results showed that low concentration inactive or active PPIase was able to improve the refolding yields, while high concentration PPIase decreased the CK reactivation yields. Aggregation was inhibited by inactive or active PPIase, and completely suppressed at 32 or 80 times the CK concentration (2.7 microM). However, alkylated PPIase was not able to prevent CK aggregation. In addition, the ability of inactive PPIase to affect CK reactivation and prevent CK aggregation was weaker than that of active PPIase. These results indicate that PPIase interacted with the early folding intermediates of CK, thus preventing their aggregation in a concentration-dependent manner. PPIase exhibited chaperone-like activity during CK refolding. The results also suggest that the isomerase activity of PPIase was independent of the chaperone activity, and that the proper molar ratio was important for the chaperone activity of PPIase. The cysteine residues of PPIase may be a peptide binding site, and may be an essential group for the chaperone function.     

Capture of monomeric refolding intermediate of human muscle creatine kinase

Human muscle creatine kinase (CK) is an enzyme that plays an important physiological role in the energy metabolism of humans. It also serves as a typical model for studying refolding of proteins. A study of the refolding and reactivation process of guanidine chloride-denatured human muscle CK is described in the present article. The results show that the refolding process can be divided into fast and slow folding phases and that an aggregation process competes with the proper refolding process at high enzyme concentration and high temperature. An intermediate in the early stage of refolding was captured by specific protein molecules: the molecular chaperonin GroEL and alpha(s)-casein. This intermediate was found to be a monomer, which resembles the "molten globule" state in the CK folding pathway. To our knowledge, this is the first monomeric intermediate captured during refolding of CK. We propose that aggregation is caused by interaction between such monomeric intermediates. Binding of GroEL with this intermediate prevents formation of aggregates by decreasing the concentration of free monomeric intermediates, whereas binding of alpha(s)-casein with this intermediate induces more aggregation.     

Identification of equilibrium and kinetic intermediates involved in folding of urea-denatured creatine kinase

The unfolding transition and kinetic refolding of dimeric creatine kinase after urea denaturation were monitored by intrinsic fluorescence and far ultraviolet circular dichroism. An equilibrium intermediate and a kinetic folding intermediate were identified and characterized. The fluorescence intensity of the equilibrium intermediate is close to that of the unfolded state, whereas its ellipticity at 222 nm is about 50% of the native state. The transition curves measured by these two methods are therefore non-coincident. The kinetic folding intermediate, formed during the burst phase of refolding under native-like conditions, possesses 75% of the native secondary structure, but is mostly lacking in native tertiary structure. In moderate concentrations of urea, only the initial, rapid change in fluorescence intensity or negative ellipticity is observed, and the final state values do not reach the equivalent unfolding values. The unfolding and refolding transition curves measured under identical conditions are non-coincident within the transition from intermediate to fully unfolded state. It is observed by SDS-PAGE that disulfide bond-linked dimeric or oligomeric intermediates are formed in moderate urea concentrations, especially in the refolding reaction. These rapidly formed, soluble intermediates represent an off-pathway event that leads to the hysteresis in the refolding transition curves.     

Proline inhibits aggregation during protein refolding

The in vitro refolding of hen egg-white lysozyme is studied in the presence of various osmolytes. Proline is found to prevent aggregation during protein refolding. However, other osmolytes used in this study fail to exhibit a similar property. Experimental evidence suggests that proline inhibits protein aggregation by binding to folding intermediate(s) and trapping the folding intermediate(s) into enzymatically inactive, "aggregation-insensitive" state(s). However, elimination of proline from the refolded protein mixture results in significant recovery of the bacteriolytic activity. At higher concentrations (>1.5 M), proline is shown to form loose, higher-order molecular aggregate(s). The supramolecular assembly of proline is found to possess an amphipathic character. Formation of higher-order aggregates is believed to be crucial for proline to function as a protein folding aid. In addition to its role in osmoregulation under water stress conditions, the results of this study hint at the possibility of proline behaving as a protein folding chaperone.     

Anion-induced folding of rabbit muscle pyruvate kinase: existence of multiple intermediate conformations at low pH

Structural and functional characteristics of rabbit muscle pyruvate kinase (PK), a tetrameric enzyme having identical subunits, were investigated under neutral as well as acidic conditions by using enzymatic activity measurements and a combination of optical methods, such as circular dichroism, fluorescence, and ANS binding. At low pH and low ionic strength, pyruvate kinase exists in a partially unfolded state (UA state) retaining half of the secondary structure and no tertiary interactions along with a strong binding to the hydrophobic dye, ANS. Addition of anions, like NaCl, KCl, and Na2SO4, to the acid-unfolded state induces refolding, resulting structural propensities similar to that of native tetramer. When anion concentration exceeds a critical limit (0.7 M KCl), a sudden loss of secondary structure and decrease in fluorescence intensity with a redshift in the emission maximum are seen which may be due to the aggregation of the protein, probably due to the intermolecular association. The anion-refolded state is more stable than the UA state, and its stability is nearly equal to that of native protein toward chemical-induced unfolding by Gu-HCl and urea. Moreover, at low concentrations, Gu-HCl behaves like an anion, by inducing refolding of the acid-unfolded state with structural features equivalent to that of native molecule. These observations support a model of protein folding where certain conformations of low free energy prevail and are populated under non-native conditions with different stability.     

Aggregation and folding of recombinant human creatine kinase

The processes of aggregation and refolding of recombinant human creatine kinase (rHCK) were studied. Most of the rHCK expressed in E. coli was present in the insoluble traction and it could be solubilized in 6 M urea solution. Unfolding of rHCK in 6 M urea showed biphasic kinetic courses (kappa1 = 6.5 x 10(-3) s(-1); kappa2 = 0.54 x 10(-3) s(-1)) as observed by maximum fluorescence wavelength change. During refolding of the rHCK dissolved in urea, significant aggregation was noticed following first-order kinetics. Aggregation rate constants were influenced by the concentration of NaCl, which increased the difference in transition-free energy (deltadeltaG), showing that stabilization of folding intermediates by NaCl could efficiently reduce the formation of insoluble aggregates. Formations of aggregate were also reduced by adjusting temperature, pH, and concentration of rHCK. Refolding of rHCK under the optimized condition which prevented the aggregation also showed multi-kinetic phases (kappa1 = 3.0 x 10(-3) s(-1); kappa2 = 0.64 x 10(-3) s(-1)). Under optimized conditions applied in this study, rHCK could correctly refold retrieving the high specific enzymatic activity.     

Effect of Zn2+ during reactivation and refolding of urea-denatured creatine kinase

The courses of refolding and reactivation of urea-denatured creatine kinase (CK) (ATP:creatine N-phosphotrans-ferase, EC 2.7.3.2) have been studied in the absence and presence of zinc ions. The presence of Zn²⁺ at low concentrations blocks the reactivation and refolding of urea-denatured CK and keeps it in a partially folded state. The partially folded state proved to be a monomeric state which resembles the molten globule state in the CK folding pathway. During refolding in the presence of Zn²⁺ , creatine kinase forms aggregates with the aggregation dependent on zinc concentration and temperature. In the presence of EDTA, the partially folded creatine kinase can be reactivated and refolded following a biphasic course, suggesting the existence of a monomeric intermediate during the refolding of CK. The results also suggest that low concentrations of zinc ions might be toxic to some proteins such as creatine kinase by disrupting their proper folding.     

Effects of osmolytes on human brain-type creatine kinase folding in dilute solutions and crowding systems

The effects of osmolytes on the unfolding and refolding process of recombinant human brain-type creatine kinase (rHBCK) were comparatively, quantitatively studied in dilute solutions and macromolecular crowding systems (simulated by 100g/L polyethylene glycol 2000), respectively. The results showed that the osmolytes, including glycerol, sucrose, dimethylsulfoxide, mannitol, inositol, and xylitol, could both protect the rHBCK from denaturation induced by 0.8M GdnHCl and aid in the refolding of denatured-rHBCK in macromolecular crowding systems. When we examined the effects of sucrose and xylitol on the parameters of residual activity, reaction kinetics and intrinsic fluorescence of rHBCK during unfolding, it was found that the protecting effects of osmolytes in a macromolecular crowding system were more significant compared with those in a dilute solution, which resulted in more residual activities, protected the conformational changes and greatly decreased the rates of both the fast and slow tracks. Regarding the effects of glycerol, sucrose and mannitol on the denatured-rHBCK refolding parameters of refolding yield, reaction kinetics and aggregation, the results indicated that the osmolytes could alleviate the aggregation of rHBCK during refolding in both dilute solutions and macromolecular crowding systems, and the refolding yields and reaction rates under macromolecular crowding environment could be increased by the addition of osmolytes, though higher yields were obtained in the dilute solution. For further insight, osmolyte docking simulations and rHBCK denaturation were conducted successfully and confirmed our experimental results. The predictions based on the docking simulations suggested that the deactivation of guanidine may be blocked by osmolytes because they share common binding sites on rHBCK, and the higher number of interactions with rHBCK by osmolytes than guanidine may be one of the causes of rHBCK refolding. In brief, the additive effects of the exclusive volume effect from the macromolecular crowding system and the osmophobic effects from the osmolytes resulted in better performance of the osmolytes in a macromolecular crowding system, which also led to a better understanding of protein folding in the intracellular environment.     

An analysis of the reassembly of denatured creatine kinase from monkey brain

Creatine kinase isolated from monkey brain was characterized with respect to denaturation/inactivation and renaturation/reactivation/reassociation in order to determine the mechanism of reassembly. Enzyme unfolded in 8 M urea exhibits several characteristics of denatured protein: complete loss of enzymatic activity, decrease in intrinsic fluorescence with a red shift in the emission maximum and loss of circular dichroism at 220 nm. The renatured protein reassembles to its apparently native condition as judged by these criteria, but small differences of uncertain origin persist. Dependence of activity and fluorescence on denaturant concentration indicate that inactivation is more sensitive to urea than is unfolding; spectral changes at the intermediate urea concentrations suggest formation of aggregated protein. Upon dilution, enzyme previously exposed to 8 M urea for 40 min regains 70-80% native activity, independent of protein concentration over the range of 0.56-160 nM. Reactivation kinetics, measured using the assay mixture with and without trypsin, are independent of protein concentration, and are adequately described by a single rate constant, 3.2 X 10(-3) s-1 and 4.2 X 10(-3) s-1, respectively. Reactivation is completed 20-30 min after initiation of renaturation. Fluorescence changes during refolding are at least biphasic, exhibiting a rapid increase, then a slow decrease completed at approximately 15-20 min after initiating refolding. Reassociation is measured by competitive hybridization between dimerizing B subunits and M subunits to form MB heterodimer. The time dependent decay in heterodimer formation during competitive dimerization shows that reassociation is completed between 60 and 90 min after initiation of reassembly. These results indicate that the brain isozyme of creatine kinase, like the muscle form, is composed of subunits which do not require association for expression of catalytic activity. Furthermore, a comparison of spectral data and susceptibility to trypsin inactivation between the muscle and brain isozymes supports previous suggestions that in the native state, the brain isozyme is a conformationally looser, more open protein.     

Fluorescence analysis of denaturation and reassembly of dansylated creatine kinase

Upon exposure of rabbit muscle creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) that has been dansylated at the two reactive lysines to 8 M urea, the maximum emission of the extrinsic fluorophore shifts 4 nm towards the blue, this being accompanied by a small decrease in intensity. The fluorescence emission and excitation spectra of the reassembled and native proteins are the same. Denaturation is accompanied by a rapid decrease in fluorescence which is complete in 10 s. This suggests that denaturation is accompanied by an early disorganization at the catalytic center, where the reactive lysines are located. Reassembly is associated with a rapid increase in dansyl fluorescence followed by a slower decrease that is complete in 6 min. Since reactivation is not complete until 20 min, minor additional structural changes are needed for the reacquisition of catalytic activity. The intrinsic protein fluorescence (eight tryptophans per dimer) of dansylated creatine kinase is approximately 60% less than that of the unlabelled enzyme, which may be attributed to resonance energy transfer, indicating that the reactive lysine is located near one or more of the tryptophans. A more limited quenching of intrinsic fluorescence is observed when dansylated creatine kinase is exposed to 8 M urea. Reassembly, monitored by a decrease in intrinsic fluorescence, reveals that the dansylated protein achieves its final fluorescence after 18 min of renaturation compared with 30 min for unlabelled enzyme. The powerful quenching by the dansyl group may limit the ability to monitor changes in the tryptophan environment. Kinetics of fluorescence polarization changes during denaturation are consistent with a mechanism involving rapid dissociation, followed by a subunit disorganization and possible aggregation. Reassembly would appear to involve first a refolding of the disorganized monomers and subsequent association. These results correspond to our previous observations that subunit renaturation precedes dimerization.     

Folding pathway for partially folded rabbit muscle creatine kinase.

Rabbit muscle creatine kinase (CK) was modified by 5,5'-dithio-bis(2-nitrobenzoic acid) accompanied by 3 M guanidine hydrochloride denaturation to produce a partially folded state with modified thiol groups. The partially folded CK was in a monomeric state detected by size exclusion chromatography, native-polyacrylamide gel electrophoresis, circular dichroism, and intrinsic fluorescence studies. After dithiothreitol (DTT) treatment, about 70% CK activity was regained with a two-phase kinetic course. Rate constants calculated for regaining of activity and refolding were compared with those for CK modified with various treatments to show that refolding and recovery of activity were synchronized. To further characterize the partially folded CK state and its folding pathway, the molecular chaperone GroEL was used to evaluate whether it can bind with partly folded CK during refolding, and 1-anilinonaphthalene-8-sulfonate was used to detect the hydrophobic surface of the monomeric state of CK. The monomeric state of CK did not bind with GroEL, although it had a larger area of hydrophobic surface relative to the native state. These results may provide different evidence for the structural requirement of GroEL recognition to the substrate protein compared with previously reported results that GroEL bound with substrate proteins mainly through hydrophobic surface. The present study provides data for a monomeric intermediate trapped by the modification of the SH groups during the refolding of CK. Schemes are given for explaining both the partial folding CK pathway and the refolding pathway.     

Aggregation of creatine kinase during refolding and chaperonin-mediated folding of creatine kinase

The course of refolding and reactivation of urea-denatured creatine kinase (ATP; creatine N-phosphotransferase, EC 2.7.3.2) has been studied in the absence and presence of molecular chaperonin GroEL. The enzyme was denatured in Tris--HCl buffer containing 6 M urea for 1 h. In the refolding studies, the denatured enzyme was diluted 60-fold into the same buffer containing GroEL or not for activity, turbidity, fluorescence measurements and polyacrylamide gel electrophoresis. The results show that the reactivation process is dependent of creatine kinase concentration in the concentration range 2.5--4 microM. The levels of activity recovery decrease with increasing enzyme concentration because of the formation of wrong aggregates. The molecular chaperonin GroEL can bind the refolding intermediate of creatine kinase and thus prevent the formation of wrong aggregates. This intermediate is an inactive dimeric form that is in a conformation resembling the 'molten globule' state.     

In vitro unfolding,refolding, and polymerization of human gammaD crystallin,a protein involved in cataract formation

Human gammaD crystallin (HgammaD-Crys), a major protein of the human eye lens, is a primary component of cataracts. This 174-residue primarily beta-sheet protein is made up of four Greek keys separated into two domains. Mutations in the human gene sequence encoding HgammaD-Crys are implicated in early-onset cataracts in children, and the mutant protein expressed in Escherichia coli exhibits properties that reflect the in vivo pathology. We have characterized the unfolding, refolding, and competing aggregation of human wild-type HgammaD-Crys as a function of guanidinium hydrochloride (GuHCl) concentration at neutral pH and 37 degrees C, using intrinsic tryptophan fluorescence to monitor in vitro folding. Wild-type HgammaD-Crys exhibited reversible refolding above 1.0 M GuHCl. The GuHCl unfolded protein was more fluorescent than its native counterpart despite the absence of metal or ion-tryptophan interactions. Aggregation of refolding intermediates of HgammaD-Crys was observed in both equilibrium and kinetic refolding processes. The aggregation pathway competed with productive refolding at denaturant concentrations below 1.0 M GuHCl, beyond the major conformational transition region. Atomic force microscopy of samples under aggregating conditions revealed the sequential appearance of small nuclei, thin protofibrils, and fiber bundles. The HgammaD-Crys fibrous aggregate species bound bisANS appreciably, indicating the presence of exposed hydrophobic pockets. The mechanism of HgammaD-Crys aggregation may provide clues to understanding age-onset cataract formation in vivo.     

Refolding kinetics of denatured-reduced lysozyme in the presence of folding aids

The refolding kinetic behavior of denatured-reduced lysozyme in the presence of folding aids (acetamide, acetone, thiourea, L-arginine or glycerol) was studied utilizing a simplified model describing the competition between first-order folding reaction and third-order aggregation. It was found that the protein folding aids could be categorized into two groups. One of them at proper concentrations, such as acetamide, acetone, thiourea and L-arginine, stabilized unfolded protein or folding intermediates. In the presence of these additives, the folding rate decreased with increasing their concentration, and there existed a concentration where the aggregation rate constant was minimized. So, there was an optimum concentration for the folding aids to produce a high yield. The other group was protein stabilizers such as glycerol. In the presence of this kind of folding aids, both the refolding rate and yield were enhanced by increasing their concentration to a proper value. Moreover, their effect on improving protein refolding was additive to those of the first group. So the cooperative application of the two kinds of folding aids could result in favorable refolding rate and yield of protein.