Cis-trans isomerization is rate-determining in the reactivation of denatured human carbonic anhydrase II as evidenced by proline isomerase.

The refolding of human carbonic anhydrase II is a sequential process. The slowest step involved is the recovery of enzymic activity (t1/2 = 9 min). Kinetic data from 'double-jump' measurements indicate that proline isomerization might be rate determining in the reactivation of the denatured enzyme. Proof of this is provided by the effect of proline isomerase on the reactivation kinetics: the presence of isomerase during reactivation lowers the half-time of the reaction to 4 min, and inhibition of proline isomerase completely abolishes this kinetic effect. A similar acceleration of the refolding process by proline isomerase is also observed for bovine carbonic anhydrase II, in contrast to what has previously been reported. In human carbonic anhydrase II there are two cis-peptidyl-Pro bonds at Pro30 and Pro202. Two asparagine single mutants (P30N and P202N) and a glycine double mutant (P30G/P202G) were constructed to investigate the role of these prolines in the rate limitation of the reactivation process. Both in the presence and absence of PPIase the P202N mutant behaved exactly like the unmutated enzyme. Thus, cis-trans isomerization of the Pro202 cis-peptidyl bond is not rate determining in the reactivation process. The mutations at position 30 led to such extensive destabilization of the protein that the refolding reaction could not be studied.     

Sequential mechanism of refolding of carbonic anhydrase B

The kinetics of refolding of bovine carbonic anhydrase B was studied by a variety of methods over a wide range of times (from milliseconds to hours). It has been shown that protein refolding proceeds through three stages. At the first stage (t1/2 approximately equal to 0.03 s) hydrophobic clusters and a compact state of the chain are formed. At the second stage (t1/2 approximately equal to 140 s) hydrophobic clusters are desolvated and the rigid native-like hydrophobic core is formed. At the third stage (t1/2 approximately equal to 600 s) the native active protein is formed.     

Trigger factor-assisted folding of bovine carbonic anhydrase II

Spontaneous refolding of GdnHCl denatured bovine carbonic anhydrase II (BCA II) shows at least three phases: a burst phase, a fast phase, and a slow phase. The fast and slow phases are both controlled by proline isomerization. However, we find that in trigger factor (TF)-assisted BCA II folding, only the fast phase is catalyzed by wild-type TF, suggesting that certain proline residues are accessible in folding intermediates. The refolding yields of BCA II assisted by wild-type TF and TF mutants which lack PPIase activity are about the same, which provides further experimental evidence that the PPIase and chaperone activities of TF are independent. The binding of TF to folding intermediates during BCA II refolding was characterized by chemical crosslinking and Western blotting. A scheme for TF-assisted BCA II folding is proposed and the possible role of the TF dimer as a "binding" chaperone in vivo is discussed.     

A nonessential role for Arg 55 in cyclophilin18 for catalysis of proline isomerization during protein folding

The protein folding process is often in vitro rate‐limited by slow cis‐trans proline isomerization steps. Importantly, the rate of this process in vivo is accelerated by prolyl isomerases (PPIases). The archetypal PPIase is the human cyclophilin 18 (Cyp18 or CypA), and Arg 55 has been demonstrated to play a crucial role when studying short peptide substrates in the catalytic action of Cyp18 by stabilizing the transition state of isomerization. However, in this study we show that a R55A mutant of Cyp18 is as efficient as the wild type to accelerate the refolding reaction of human carbonic anhydrase II (HCA II). Thus, it is evident that the active‐site located Arg 55 is not required for catalysis of the rate‐limiting prolyl cis‐trans isomerization steps during the folding of a protein substrate as HCA II. Nevertheless, catalysis of cis‐trans proline isomerization in HCA II occurs in the active‐site of Cyp18, since binding of the inhibitor cyclosporin A abolishes rate acceleration of the refolding reaction. Obviously, the catalytic mechanisms of Cyp18 can differ when acting upon a simple model peptide, four residues long, with easily accessible Pro residues compared with a large protein molecule undergoing folding with partly or completely buried Pro residues. In the latter case, the isomerization kinetics are significantly slower and simpler mechanistic factors such as desolvation and/or strain might operate during folding‐assisted catalysis, since binding to the hydrophobic active site is still a prerequisite for catalysis.     

Multiparameter kinetic study on the unfolding and refolding of bovine carbonic anhydrase B

The kinetics of unfolding and refolding of bovine carbonic anhydrase B by guanidinium chloride have been studied by simultaneously monitoring several spectroscopic parameters, each of which reflects certain unique conformational features of the protein molecule. In the present report, far-UV circular dichroism (CD) was used to follow the secondary structural change, UV difference absorption was used to follow the exposure or burying of aromatic amino acid residues, and near-UV CD was used to follow tertiary structural changes during unfolding and refolding. The unfolding is described by two unimolecular rate processes, and refolding is described by three unimolecular rate processes. The minimum number of conformational species involved in the mechanism is five. The refolding of the protein followed by the above three parameters indicates that the process consists of an initial rapid phase in which the random-coiled protein is converted to an intermediate state(s) having secondary structure comparable to that of the native protein. This is followed by the burying of the aromatic amino acid residues to form the interior of the protein molecule. Subsequently, the protein molecule acquires its tertiary structure and folds into a unique conformation with the formation of aromatic clusters.     

Folding of chymotrypsin inhibitor 2. 2. Influence of proline isomerization on the folding kinetics and thermodynamic characterization of the transition state of folding

The refolding of chymotrypsin inhibitor 2 (CI2) is, at least, a triphasic process. The rate constants are 53 s-1 for the major phase (77% of the total amplitude) and 0.43 and 0.024 s-1 for the slower phases (23% of the total amplitude) at 25 degrees C and pH 6.3. The multiphase nature of the refolding reaction results from heterogeneity in the denatured state because of proline isomerization. The fast phase corresponds to the refolding of the fraction of protein that has all its prolines in a native trans conformation in the denatured state. It is not catalyzed by peptidyl-prolyl isomerase. The rate-limiting step of folding for the slower phases, however, is proline isomerization, and they are both catalyzed by peptidyl-prolyl isomerase. The slowest phase has properties consistent with a process involving proline isomerization in a denatured state. In particular, the activation enthalpy is large, 16 kcal mol-1 K-1, and the rate is independent of guanidinium chloride concentration ([GdnHCl]). In comparison, the intermediate phase shows properties consistent with a process involving proline isomerization in a partially structured state. The activation enthalpy is small, 8 kcal mol-1 K-1, and the rate has a strong dependence on [GdnHCl]. Temperature dependences of the rate constants for unfolding and for the fast refolding phase, both in the absence and in the presence of GdnHCl, were used to characterize the thermodynamic nature of the transition state and its relative exposure to solvent. The Eyring plot for unfolding is linear, indicating that there is relatively little change in heat capacity between native state and transition state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of hemoglobin in proton transfer to the active site of carbonic anhydrase.

The binding of bovine oxyhemoglobin to bovine carbonic anhydrase with a dissociation constant between 10(-5) and 10(-7) M has been determined by countercurrent distribution using aqueous, biphasic polymer systems. This result provides an explanation for the very efficient proton transfer between hemoglobin and carbonic anhydrase, a transfer which enhances the catalytic activity of carbonic anhydrase as measured by 18O exchange between bicarbonate and water at chemical equilibrium (Silverman, D. N., Tu, C. K., and Wynns, G. C. (1978) J. Biol. Chem, 253, 2563-2567). Two rate constants describing 18O exchange activity of carbonic anhydrase at pH 7.5 show saturation behavior when plotted against hemoglobin concentration consistent with a dissociation constant of 2.5 X 10(-6) M between bovine hemoglobin and carbonic anhydrase. Interpretation of these rate constants in terms of a two-step model for 18O exchange indicates that hemoglobin enhances the rate of exchange from carbonic anhydrase of water containing the oxygen abstracted from bicarbonate, but does not affect the catalytic interconversion of CO2 and HCO3- at chemical equilibrium.     

Effect of multiple prolyl isomerization reactions on the stability and folding kinetics of the notch ankyrin domain: experiment and theory

Studies on the folding kinetics of the Notch ankyrin domain have demonstrated that the major refolding phase is slow, the minor refolding phase is limited by the isomerization of prolyl peptide bonds, and that unfolding is multiexponential. Here, we explore the relationship between prolyl isomerization and folding heterogeneity using a combination of experiment and simulation. Proline residues were replaced with alanine, both singly and in various combinations. These destabilizing substitutions combine to eliminate the minor refolding phase, although unfolding heterogeneity persists even when all seven proline residues are replaced. To test whether prolyl isomerization influences the major refolding phase, we modeled folding and prolyl isomerization as a system of sequential reactions. Simulations that use rate constants of the major folding phase of the Notch ankyrin domain to represent intrinsic folding indicate that even with seven prolyl isomerization reactions, only two significant phases should be observed, and that the fast observed phase provides a good approximation of the intrinsic folding in the absence of prolyl isomerization. These results indicate that the major refolding phase of the Notch ankyrin domain reflects an intrinsically slow folding transition, rather than coupling of fast folding events with slow prolyl isomerization steps. This is consistent with the observation that the single observed refolding phase of a construct in which all proline residues are replaced remains slow. Finally, the simulation fails to produce a second unfolding phase at high urea concentrations, indicating that prolyl isomerization does not play a role in the three-state mechanism that leads to this heterogeneity.     

Improved protein refolding using hollow-fibre membrane dialysis

We have used a cellulose acetate, hollow-fibre (HF) ultrafiltration membrane to refold bovine carbonic anhydrase, loaded into the lumen space, by removing the denaturant through controlled dialysis via the shell side space. When challenged with GdnHCl-denatured carbonic anhydrase, 70% of the loaded protein reptated through the membrane into the circulating dialysis buffer. Reptation occurred because the protein, in its fully unfolded configuration, was able to pass through the pores. The loss of carbonic anhydrase through the membrane was controlled by the dialysis conditions. Dialysis against 0.05 M Tris-HCl for 30 min reduced the denaturant around the protein to a concentration that allowed the return of secondary structure, increasing the hydrodynamic radius, thus preventing protein transmission. Under these conditions a maximum of 42% of carbonic anhydrase was recovered (from a starting concentration of 5 mg/mL) with 94% activity. This is an improvement over refolding carbonic anhydrase by simple batch dilution, which gave a maximum reactivation of 85% with 35% soluble protein yield. The batch refolding of carbonic anhydrase is very sensitive to temperature; however, during HF refolding between 0 and 25 degrees C the temperature sensitivity was considerably reduced. In order to reduce the convection forces that give rise to aggregation and promote refolding the dialyzate was slowly heated from 4 to 25 degrees C. This slow, temperature-controlled refolding gave an improved soluble protein recovery of 55% with a reactivation yield of 90%. The effect of a number of additives on the refolding system performance were tested: the presence of PEG improved both the protein recovery and the recovered activity from the membrane, while the detergents Tween 20 and IGEPAL CA-630 increased only the refolding yield.     

The effect of methanol and temperature on the kinetics of refolding of ribonuclease A

Unfolded ribonuclease A consists of 20% fast refolding (Uf) and 80% slow refolding material (Us). The latter consists of at least two different forms which refold at different rates. We have used absorbance and fluorescence spectrophotometry to compare the kinetics of refolding in aqueous and aqueous-methanol solutions. At 1 degree C and pH 3.0, the addition of increasing concentrations of methanol (to 50%, v/v) had negligible effect on the rates and amplitudes of the slow refolding Us states. The effect of temperature on the two slow phases of refolding was determined in 35 and 50% methanol. From Arrhenius plots the energies of activation were found to be in the vicinity of 20 kcal/mol for both processes. The results suggest that both slow phases correspond to proline isomerization, and that the presence of methanol does not significantly perturb the overall refolding process. It is possible that the faster of the slow refolding phases corresponds to the isomerization of a proline residue which is trans in the folded native state but which undergoes extensive isomerization to the cis conformation in the unfolded state.     

Osmolyte effects on kinetics of FKBP12 C22A folding coupled with prolyl isomerization

Unfolding and refolding kinetics of human FKBP12 C22A were monitored by fluorescence emission over a wide range of urea concentration in the presence and absence of protecting osmolytes glycerol, proline, sarcosine and trimethylamine-N-oxide (TMAO). Unfolding is well described by a mono-exponential process, while refolding required a minimum of two exponentials for an adequate fit throughout the urea concentration range considered. The bi-exponential behavior resulted from complex coupling between protein folding, and prolyl isomerization in the denatured state in which the urea-dependent rate constant for folding was greater than, equal to, and less than the rate constants for prolyl isomerization within the urea concentration range of zero to five molar. Amplitudes and the observed folding and unfolding rate constants were fitted to a reversible three-state model composed of two sequential steps involving the native state and a folding-competent denatured species thermodynamically linked to a folding-incompetent denatured species. Excellent agreement between thermodynamic parameters for FKBP12 C22A folding calculated from the kinetic parameters and those obtained directly from equilibrium denaturation assays provides strong support for the applicability of the mechanism, and provides evidence that FKBP12 C22A folding/unfolding is two-state, with prolyl isomer heterogeneity in the denatured ensemble. Despite the chemical diversity of the protecting osmolytes, they all exhibit the same kinetic behavior of increasing the rate constant of folding and decreasing the rate constant for unfolding. Osmolyte effects on folding/unfolding kinetics are readily explained in terms of principles established in understanding osmolyte effects on protein stability. These principles involve the osmophobic effect, which raises the Gibbs energy of the denatured state due to exposure of peptide backbone, thereby increasing the folding rate. This effect also plays a key role in decreasing the unfolding rate when, as is often the case, the activated complex exposes more backbone than is exposed in the native state.     

An approach for the assessment of the order of disruption of the elements of protein structure upon protein unfolding: A study of carbonic anhydrase B

An experimental approach named μ-analysis has been developed in order to elucidate the sequence of the loss of ordered structure by elements of a protein during the denaturation of the molecule. This approach is applicable for the analysis of proteins that fold (unfold) in a multistep process that involve the formation (destruction) of a range of intermediate states. The concept of the approach consists in systematic analysis of mutagenized forms of the protein with point substitutions of hydrophobic amino-acid residues and additional cysteine bridges. Importantly, the substitutions of the amino-acid residues must be localized to the same structural elements of the protein. Point substitutions of hydrophobic amino-acid residues mainly provide information on the structural elements of the protein that are disrupted at the final stages of protein denaturation. The addition of cysteine bridges to the surface of the protein molecule allows investigation of structural elements of the protein that are the first to unfold upon protein denaturation. Calorimetric studies of non-equilibrium melting of bovine carbonic anhydrase B yielded information on the rate constants of the unfolding of ten mutant forms of the protein. The analysis of the effects of mutations on the rates of different stages of protein unfolding allowed for elucidation of the order of disruption of structural elements of carbonic anhydrase B upon thermal denaturation.     

The carbon dioxide hydration activity of brush-border carbonic anhydrase from the dog kidney

The steady-state kinetic parameters for the hydration of CO₂ catalyzed by membrane-bound carbonic anhydrase from the renal brush-border of the dog are compared with the same parameters for water-soluble bovine erythrocyte carbonic anhydrase. For the membrane-bound enzyme, the turnover number k_cat is 6.5 × 10⁵ s^?1 and the Michaelis constant is 7.5 mm for CO₂ hydration at pH 7.4 and 25 °C. The corresponding constants for bovine carbonic anhydrase under these conditions are 6.3 × 10⁵ s^?1 and 15 mm (Y. Pocker and D.W. Bjorkquist (1977)Biochemistry16, 5698–5707). The rate constant for the transfer of a proton between carbonic anhydrase and buffer was determined from the dependence of the catalytic rate on the concentration of the buffers imidazole and N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (Hepes); the value of 2 × 10⁸m^?1s^?1 describes this constant for both forms of carbonic anhydrase at pH 7.4. Furthermore, the pH dependence of the initial velocity of hydration of CO₂ in the range of pH 6.5 to 8.0 is identical for the membrane-bound and soluble enzyme at low buffer concentration (1–2 mm imidazole). We conclude that the membrane plays no detectable role in affecting the CO₂ hydration activity and that the active site of the renal, membrane-bound carbonic anhydrase is exposed to the aqueous phase.     

Purification and characterization of carbonic anhydrase from the saliva of the rat

Carbonic anhydrase purified from the saliva of the rat had kinetic properties identical with those of carbonic anhydrase II from rat red cells, but its molecular properties were distinctly different from the type II isozyme. Kinetic parameters were measured under steady state conditions by stopped-flow spectrophotometry and under equilibrium conditions by an 18O exchange method. The turnover number kcat for hydration of CO2 was 6.5 X 10(4) s-1 and the Michaelis constant was 4.2 mM at pH 7.5 and 25 degrees C, values which are equal to the steady state constants for red cell carbonic anhydrase II from the rat. Inhibition of the salivary isozyme by sulfanilamide (Ki = 3.7 microM) was nearly as efficient as inhibition of the erythrocyte isozyme II (Ki = 1.1 microM). The molecular weight for the salivary isozyme was 46,000 and the isoelectric point was 5.5. Salivary carbonic anhydrase had high mannose oligosaccharide components as measured by concanavalin A binding. The amino acid composition for the salivary isozyme was not similar to rat type II, but it was similar to that reported for membrane-bound carbonic anhydrase from bovine lung (Whitney, P.L., and Briggle, T.V. (1982) J. Biol. Chem. 257, 12056-12059). These observations suggest to us that salivary carbonic anhydrase is a secretory product.     

Catalysis of proline isomerization during protein-folding reactions

The enzyme peptidylprolyl cis-trans isomerase (PPI) is known to catalyze proline isomerization in short proline-containing peptides. If PPI can be shown to generally catalyze isomerization of proline residues in proteins, then it would be a valuable diagnostic reagent for recognition of isomerization, which has proven to be extremely difficult to characterize by other methods. In this study, the catalytic effect of PPI on the slow refolding reactions of seven different proteins has been studied, and in only two cases (RNase T1 and cytochrome c) could significant catalysis be seen. PPI also caused no enhancement in the rate for the 'subtle' conformational changes of native concanavalin A or native Fragment I of prothrombin, which have been suggested to be rate-limited by proline isomerization. There was a small effect of PPI observed for the generation of native RNAase A from the fully-reduced form when the glutathione concentration was low. The conclusion from these studies is that PPI can weakly catalyze some protein processes which are rate-limited by proline isomerization, but probably exhibits no measureable catalysis toward others. This somewhat limits the usefulness of PPI as a diagnostic reagent for proline isomerization.     

Inhibition of aggregation by media selection,sample loading and elution in size exclusion chromatographic refolding of denatured bovine carbonic anhydrase B

The effects of gel media, sample application and elution flowrate on the activity recovery and aggregation in refolding of bovine carbonic anhydrase B (CAB) by size exclusion chromatography (SEC) were investigated. Variation in aggregation was demonstrated visibly by the comparison of refolding profiles under different operating conditions. Some principles with regard to practical application were proposed. Meanwhile, the analysis of relationship between peak resolution and activity recovery provided evidences for the mechanism of size exclusion chromatography protein refolding.     

Coupled kinetic traps in cytochrome c folding: His-heme misligation and proline isomerization

The effect of His-heme misligation on folding has been investigated for a triple mutant of yeast iso-2 cytochrome c (N26H,H33N,H39K iso-2). The variant contains a single misligating His residue at position 26, a location at which His residues are found in several cytochrome c homologues, including horse, tuna, and yeast iso-1. The amplitude for fast phase folding exhibits a strong initial pH dependence. For GdnHCl unfolded protein at an initial pH<5, the observed refolding at final pH 6 is dominated by a fast phase (tau(2f)=20 ms, alpha(2f)=90 %) that represents folding in the absence of misligation. For unfolded protein at initial pH 6, folding at final pH 6 occurs in a fast phase of reduced amplitude (alpha(2f) approximately 20 %) but the same rate (tau(2f)=20 ms), and in two slower phases (tau(m)=6-8 seconds, alpha(m) approximately 45 %; and tau(1b)=16-20 seconds, alpha(1b) approximately 35 %). Double jump experiments show that the initial pH dependence of the folding amplitudes results from a slow pH-dependent equilibrium between fast and slow folding species present in the unfolded protein. The slow equilibrium arises from coupling of the His protonation equilibrium to His-heme misligation and proline isomerization. Specifically, Pro25 is predominantly in trans in the unligated low-pH unfolded protein, but is constrained in a non-native cis isomerization state by His26-heme misligation near neutral pH. Refolding from the misligated unfolded form proceeds slowly due to the large energetic barrier required for proline isomerization and displacement of the misligated His26-heme ligand.     

Effects of proline mutations on the unfolding and refolding of human lysozyme: the slow refolding kinetic phase does not result from proline cis-trans isomerization

The unfolding and refolding kinetics of six proline mutants of the human lysozyme (h-lysozyme) were carried out and compared to that of the wild-type protein. Our results show that the slow refolding phase observed in the h-lysozyme refolding kinetics cannot be ascribed to proline isomerization reactions. The h-lysozyme contains two proline residues at positions 71 and 103, both in the trans conformation in the native state. The refolding kinetics of the P71G/P103G mutant, in which both prolines have been replaced by a glycine, were found to be similar to those of the wild-type protein. The same slow phase amplitude of about 10% was found for both proteins, and the slow phase rate constants were also identical within experimental error. Other mutants such as P103G or P71G, in which only one of the two prolines has been replaced by a glycine, and A47P with its three prolines, gave identical slow refolding phases. The X-ray structure analysis and scanning microcalorimetric study of each protein (Herning et al., unpublished experiments) have confirmed that none of the considered mutations affects significantly protein structure and that no major changes in protein stability were brought about by these mutations. Therefore, comparison of the properties of the mutant and wild-type proteins is legitimate. Interestingly, the refolding kinetics of the V110P mutant, in which a proline residue has been introduced at position 110 (N-terminus of an alpha-helix), were clearly triphasic. For this mutant an additional very slow phase with properties similar to those expected from the proline hypothesis was detected. Equilibrium denaturation studies were conducted for each protein, and the refolding pathway of h-lysozyme is partly presented. We also discuss the effect of proline mutations on the energetics of the folding pathway of the h-lysozyme in water.     

Proline isomerization during refolding of ribonuclease A is accelerated by the presence of folding intermediates

The trans----cis isomerization of Pro 93 was measured during refolding of bovine ribonuclease A. This isomerization is slow (tau = 500 s) under marginally stable folding conditions of 2.0 M GdmCl, pH 6, at 10 degrees C. However, it is strongly accelerated (tau = 100 s) in samples which, prior to isomerization, had been converted to a folding intermediate by a 15 s refolding pulse under strongly native conditions (0.8 M ammonium sulfate, 0 degree C). The results demonstrate that extensive folding is possible before Pro 93 isomerizes to its native cis state and that the presence of structural folding intermediates leads to a marked increase in the rate of subsequent proline isomerization.     

Polyethylene glycol enhanced refolding of bovine carbonic anhydrase B. Reaction stoichiometry and refolding model.

Polyethylene glycol (PEG) inhibited aggregation during refolding of bovine carbonic anhydrase B (CAB) through the formation of a nonassociating PEG-intermediate complex. Stoichiometric concentrations of PEG were required for complete recovery of active protein during refolding at aggregating conditions. For example, a PEG (Mr = 3350) to CAB molar ratio ([PEG]/[CAB]) of 2 was sufficient to inhibit aggregation during refolding at 1.0 mg/ml (33.3 microM) protein and 0.5 M guanidine hydrochloride. In addition, the PEG concentration required for enhancement was dependent upon the molecular weight and only molecular weights between 1000 and 8000 were effective in inhibiting aggregation. In the presence of PEG, the rate of refolding was the same as that observed for refolding without the formation of associated species. Refolding in the presence of PEG resulted in the rapid formation of a PEG complex with the molten globule first intermediate, and this PEG-intermediate complex did not aggregate. The CAB refolding kinetics in the presence of PEG were determined and used to develop a model of the PEG enhanced refolding pathway. The mathematical model was validated by independent activity measurements of CAB refolding. This model predicted that PEG enhanced refolding of CAB occurred by a specific interaction of PEG with the molten globule first intermediate to form a nonassociating complex which continued to fold at the same rate as the first intermediate. The predicted pathway and binding properties of PEG indicate that PEG enhanced refolding may be analogous to chaperonin mediated protein folding.