©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Kinetic Analysis of the Folding of Human Carbonic Anhydrase II and Its Catalysis by Cyclophilin (*)

(Received for publication, September 7, 1994; and in revised form, November 2, 1994)

Gunther Kern (1)(§) Dorothee Kern (1) Franz X. Schmid (2) Gunter Fischer (1)

From the  (1)Max-Planck-Arbeitsgruppe ``Enzymologie der Peptidbindung,'' Weinbergweg 16A, D-06120 Halle/Saale, Germany and the (2)Laboratorium für Biochemie, Universität Bayreuth, D-95440 Bayreuth, Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The kinetics of unfolding and refolding of human carbonic anhydrase II (HCAII) and its catalysis by the peptidyl-prolyl-cis/trans-isomerase cyclophilin were investigated. HCAII contains 15 trans- and 2 cis-prolyl peptide bonds, and, when long-term denatured, virtually all unfolded molecules contain non-native prolyl isomers. In unfolding these molecules (U(s)) are produced slowly in a biphasic process reflecting the isomerization of several trans-prolines and of one cis-proline. In refolding, the rapid formation of an intermediate of the molten globule type is followed by several slow prolyl isomerizations, which determine the rate of reactivation. By a short 10-s incubation in 5.0 M guanidinium chloride at 2 °C, unfolded HCAII species with all prolines still in the native conformation (U(f)) could be produced. Surprisingly, only a fraction of U(f) refolds rapidly, but the other molecules refold slowly. Evidently, some prolyl peptide bonds isomerize early in refolding, at the stage of the molten globule and as a consequence, molecules with incorrect prolyl isomers are formed in competition with the productive folding of U(f). This fraction of slow-folding molecules is strongly increased when cyclophilin is present, because it accelerates the formation of non-native prolyl isomers as long as the molecules remain in the molten globule state. Later cyclophilin catalyzes the isomerization of these prolyl peptide bonds toward the native state, which are stabilized in their conformation by further folding to the native state. This catalysis is very efficient, because only prolines that are accessible in the molten globule are involved in this sequence of isomerization and reisomerization.


INTRODUCTION

cis/trans-isomerizations of prolyl peptide bonds are slow reactions, and redundant they determine the rate of the in vitro folding of many proteins(1) . The peptidyl-prolyl-cis/trans-isomerases (EC 5.2.1.8) catalyze these isomerizations in small peptides (2) as well as in protein folding(3, 4, 5) . Two structurally unrelated classes of peptidyl-prolyl-cis/trans-isomerases have been characterized: the cyclophilins (^1)and the FK506 binding proteins. They bind tightly to the immunosuppressive drugs cyclosporin A and FK506, respectively, and this binding inhibits the isomerase activities of both peptidyl-prolyl-cis/trans-isomerases. In vivo inhibition of cyclophilin by cyclosporin A prevents the folding and secretion of transferrin (6) and retards the formation of the collagen triple helix(7) . In addition, members of the cyclophilin family promote the productive folding of Rh1 rhodopsin (8, 9) and bind to the gag protein from human immunodeficiency type 1 virus(10) . Recently, a third class of small peptidyl-prolyl-cis/trans-isomerases, the parvulins, was discovered(11) .

The efficiency as a catalyst of in vitro protein folding varies strongly, depending on the nature of the peptidyl-prolyl-cis/trans-isomerases and on the properties of the refolding protein(5, 12) . For folding of several proteins a catalysis could not be detected, even though refolding is limited in rate by prolyl isomerizations. Often, a partially ordered structure forms very rapidly during refolding and decreases the accessibility of the prolyl peptide bonds for peptidyl-prolyl-cis/trans-isomerases(3, 4, 13, 14, 15) .

Human carbonic anhydrase II (HCAII) refolds in two stages. Intermediates of the molten globule type are formed rapidly in the first stage, followed by slow reactions, which determine the overall rate of refolding. These slow reactions show properties of prolyl isomerizations and are accelerated by peptidyl-prolyl-cis/trans-isomerases(16) . Freskgard et al.(17) suggested that cytosolic porcine kidney cyclophilin 18 exerted a dual function as a peptidyl-prolyl-cis/trans-isomerases and as a chaperone in the refolding of HCAII. A reinvestigation of the refolding of HCAII indicated, however, that the observed effects of cyclophilin can be explained solely by its peptidyl-prolyl-cis/trans-isomerases activity and that it is not necessary to postulate a chaperone function for peptidyl-prolyl-cis/trans-isomerases(18) .

It was our aim here to elucidate the relationship between the rapid formation of the molten globule in the folding of HCAII and the subsequent slow prolyl isomerizations, as well as the catalysis of these steps by cyclophilin. The formation of the early folding intermediates was detected by circular dichroism, and the slow, rate-limiting steps of folding were followed by the regain of HCAII activity. We performed two different types of unfolding/refolding experiments. (^2)In the first experiments the protein was initially unfolded for 1 h at 20 °C to produce U(s) molecules with prolyl peptide bonds in thermodynamic cis/trans-equilibrium and then their role in refolding and in catalysis by cyclophilin were studied. In a second set of experiments the initial unfolding step was shortened to 10 s at 2 °C to produce U(f) molecules, which had not yet undergone prolyl isomerizations. Their refolding was also measured and compared with the folding of the U(s) molecules. Finally the duration of unfolding in the first step was varied, and the kinetics of interconversion between U(f) and U(s) in the unfolded protein were followed by the decrease in the amplitude of the fast reaction in the refolding step.


MATERIALS AND METHODS

HCAII was a gift from Dr. Uno Carlsson (Linköping University, Sweden). The molecular mass of 29,097 ± 4 Da was determined by mass spectrometry and sequencing of the N terminus delivered the sequence AHWGYGKHN. Cyclophilin was kindly provided by Dr. Kurt Lang, Boehringer Mannheim (Penzberg, Germany). Guanidinium hydrochloride (GdmHCl) and Tris buffer, ultra pure, were purchased from Schwarz/Mann. 4-Nitrophenyl acetate was from Sigma and was recrystallized from anhydrous diethyl ether to a constant melting point of 79-80 °C. All other chemicals used were analytical-grade.

Denaturation and Renaturation

The concentration of HCAII was determined spectrophotometrically using a molar extinction coefficient of = 8730 M cm(19) . HCAII (17 µM) was denatured in 5.0 M GdmHCl, 0.1 M Tris/H(2)SO(4), pH 7.5, for 1 h at 20 °C to produce long-term denatured protein. Short-term denatured HCAII was produced by incubation in the same buffer at 2 °C for 10 s. Reactivation was started by rapid 17-fold dilution with 0.1 M Tris/H(2)SO(4), pH 7.5, 20 °C to a final HCAII concentration of 1 µM. For rapid mixing the reactivation solution containing the respective cyclophilin concentration was quickly injected into the solution of the denatured protein. Reactivation kinetics and yields were determined following the esterase activity of HCAII toward 4-nitrophenyl acetate by the continuous as well as by the discontinuous assay method. The experimental data were fitted using the software Sigma Plot from Jandel Scientific (Erkrath, Germany).

Far UV-CD Measurements

CD measurements were performed in a Jasco J-710 spectropolarimeter in a thermostatted cuvette holder using 0.1-cm quartz cells. For calibration an aqueous 1 mg/ml d-camphor-10 sulfonic acid solution (20) was used. The molar ellipticity was calculated for the mean residue molecular weight in HCAII. For kinetic measurements, all solutions, vials, and pipette tips were thermostatted at 10 °C. Mixing was performed in an Eppendorf vial before the mixed solution was transferred to the cuvette. The time required for the overall process was 15 s.

Continuous and Discontinuous Reactivation Assay

All activity measurements were performed as described by Kern et al.(18) .

Double Jump Experiment

To determine the decrease of U(f) molecules as a function of denaturation time at 2 °C, renaturation was initiated by rapid dilution with Tris buffer, pH 7.5, at 20 °C after various incubation times. The amount of U(f) molecules produced during the denaturation was determined by extrapolation of the slow reaction in the resulting reactivation curves to 0-s reactivation. Reactivation was followed using the discontinuous method.


RESULTS

Spectroscopic Investigations of the Kinetics of Unfolding and Refolding of HCAII

To characterize the folding and isomerization reactions of HCAII we unfolded the native protein first by dilution to final conditions of 5.0 M GdmHCl in 0.1 M Tris-HCl, pH 7.5, and then refolding was initiated by a second dilution to 0.3 M GdmHCl in the same buffer. The refolding of HCAII was investigated using CD and fluorescence spectroscopy and an improved esterase activity assay(18) . Fig. 1A shows the far UV-CD spectra of fully denatured and native HCAII. In unfolding the changes in the CD signal at 220 nm are rapid and complete within the dead time of manual mixing (15 s) even when unfolding is performed at 2 °C (Fig. 1B). This indicates that the loss of ordered structure in the unfolding of HCAII in 5 M GdmHCl is a very rapid reaction and thus much faster than the subsequent prolyl isomerizations in the unfolded protein.


Figure 1: A, CD spectra of native (-) and denatured(- - - -) HCAII in the far UV region. The protein concentration was 6 µM in 1-mm cells for the denatured HCAII and 0.6 µM in 1-cm cells for the native HCAII. B, kinetics of denaturation (bulletbulletbullet) and renaturation (-) of HCAII. The kinetics were followed by the CD signal at 220 nm as indicated by the vertical line in A. Denaturation was initiated by 10-fold dilution of 6 µM native HCAII with 0.1 M Tris, pH 7.5, 5 M GdmHCl at 2 °C. Renaturation was started by 10-fold dilution of 6 µM HCAII, denatured in 5 M GdmHCl, 0.1 M Tris, pH 7.5, at 20 °C. The time required for manual mixing was 15 s. The protein concentration was 0.6 µM in 1-cm cells.



In refolding the secondary structure is also regained very rapidly and the CD signal of the native protein at 220 nm is restored completely within less than 15 s (Fig. 1B). A similar result was obtained when refolding was followed by the change in tryptophan fluorescence at 322 nm ( = 280 nm). Again the signal of the native protein is regained within less than 15 s (data not shown). In contrast, reactivation of HCAII is very slow, and less than 4% of the activity of the native protein is restored after 15 s of refolding under the same conditions. Apparently, the secondary and part of the tertiary structure of HCAII are formed very rapidly during refolding, and kinetic intermediates of the molten globule type are formed transiently. Such intermediates were also found in the equilibrium unfolding transitions of the human (HCAII) (21) and bovine (BCA) protein(22, 23, 24) .

The cis/trans-isomerizations of the incorrect prolines are intrinsically slow reactions and occur at the stage of these largely folded intermediates. They determine the rates of the final steps of folding, which lead to the native, catalytically active protein. For BCA it was shown that these final slow reactions are also accompanied by a change in the CD signal of the aromatic residues, in the accessibility of tryptophan residues to fluorescence quenching, and in the ^1H NMR spectra(24, 25) . CD measurements at 270 nm require protein concentrations higher than 0.1 mg/ml in the refolding experiment. However, at these concentrations HCAII aggregates.

Outline of the Folding Experiments

To study the role of prolyl isomerizations in the refolding of HCAII and the catalysis by cyclophilin we carried out two different kinds of reactivation experiments. In the first set of experiments, HCAII was incubated in 5.0 M GdmHCl at 20 °C for a long time (1 h) to unfold the protein, and, in addition, to let all prolyl isomerizations in the unfolded molecules proceed to equilibrium. This equilibrium mixture of species is called U(s). Its refolding was then investigated after a 17-fold dilution to 0.3 M GdmHCl at 20 °C in the absence and presence of cyclophilin.

In the second set of experiments the protein was exposed only briefly (for 10 s) to 5 M GdmHCl at low temperature (2 °C). During this short period of time only the unfolding reaction takes place (cf. Fig. 1), but not the slow prolyl isomerizations, and, as a consequence, unfolded molecules with the native set of prolyl isomers (U(f)) are produced by this brief unfolding pulse. Again, after dilution to native solvent conditions, their refolding and the influence of cyclophilin could be examined.

Kinetics of Reactivation of HCAII after Long-term Unfolding

As outlined above the equilibrium mixture of unfolded molecules with different prolyl isomers (U(s)) was produced by unfolding for 1 h, and then reactivation was measured in the absence and presence of 0.25-10 µM cyclophilin (Fig. 2). Under the given conditions about 80% of the original activity was regained after unfolding and refolding. Reactivation followed a complex time course, and, to a first approximation, it could be analyzed as the sum of two exponential functions. In the absence of cyclophilin, apparent rate constants of k(2) = 2.2 times 10 s and k(3) = 5 times 10 s and fractional amplitudes of A(2) = 0.27 and A(3) = 0.51 were obtained. In the presence of cyclophilin slow refolding is accelerated (Fig. 2), confirming earlier measurements(16) . The analysis of the kinetics in the presence of cyclophilin (Fig. 2) suggests that the rate of the slowest phase (k(3)) seems to be rather unaffected by cyclophilin, it does, however, lose amplitude, and A(3) decreases from 0.51 in the absence of cyclophilin to 0.11 in the presence of 10 µM cyclophilin (Table 1). The rate of the faster process (k(2)) increases moderately with increasing concentration of cyclophilin in a nonlinear fashion, and the amplitude of this reaction grows at the expense of the amplitude of the slowest process. The representation of the experimental reactivation kinetics as a sum of two exponential functions is certainly an oversimplification, and therefore a further analysis of the apparent rate constants and amplitudes is not warranted. HCAII contains 17 prolines and certainly many unfolded species exist, which may differ in the rate of refolding and in the extent of catalysis. An effect of cyclophilin on the apparent amplitudes of refolding was also observed for RNase T1(14) . The kinetic data are summarized in Table 1and show that the final reactivation yield is unaffected by cyclophilin.


Figure 2: Reactivation kinetics of long-term denatured HCAII in the absence and presence of various amounts of cyclophilin. 17 µM HCAII was denatured in 5 M GdmHCl, 0.1 M Tris/SO(4), pH 7.5, for 1 h at 20 °C. Reactivation was started by rapid 17-fold dilution with 0.1 M Tris/SO(4), pH 7.5, 20 °C in the absence (circle) and presence of 0.25 µM (), 1 µM (bullet), 2 µM (), and 10 µM () cyclophilin. The solid lines are fits according to two parallel first-order reactions. Reactivation was followed with the continuous reactivation assay. Final yields were determined with both the continuous and discontinuous assay.





Kinetics of Refolding of Short-term Denatured HCAII

Unfolded molecules of HCAII with the prolyl peptide bonds still in their native conformation (U(f)) were produced by a short 10-s unfolding pulse in 5.0 M GdmHCl at 2 °C, and their refolding was studied at 20 °C. The U(f) molecules should not contain incorrect prolyl isomers, and therefore a single rapid reactivation reaction should be observed, which is not affected by cyclophilin. The results in Fig. 3indicate, however, that the reactivation of U(f) molecules in the absence of cyclophilin is clearly a biphasic reaction. Only about 55% of the activity is regained in a rapid reaction with an apparent rate constant of k(1) = 7.3 times 10 s. This reaction is complete within 45 s, it is not observed in the reactivation of U(s) molecules (Fig. 2), and is apparently not limited by prolyl isomerizations. The remaining 25% of activity is regained very slowly, with a rate constant (k(2) = 10 s) similar to the rates of slow refolding of the long-term unfolded protein (cf. Fig. 2and Table 1). Apparently, 25-45% of molecules with incorrect prolyl isomers are present in this experiment and give rise to this slow reaction.


Figure 3: Reactivation kinetics of short-term denatured HCAII in the absence (circle) and presence (bullet) of 1 µM cyclophilin. 17 µM HCAII was denatured in 5 M GdmHCl, 0.1 M Tris/SO(4), pH 7.5, for 10 s at 2 °C and reactivation started by rapid 17-fold dilution with 0.1 M Tris/SO(4), pH 7.5, at 20 °C containing no or 1.06 µM cyclophilin, respectively. The solid lines represent fits according to two parallel first-order reactions. Reactivation was followed with both the discontinuous and continuous assay method.



These molecules could have been already formed during the 10-s unfolding pulse by some unusually rapid prolyl isomerizations, or, else, early in refolding at the stage of the molten globule intermediate by prolyl isomerizations that compete with refolding of U(f) after the jump from 5.0 M GdmHCl, 2 °C, to 0.3 M GdmHCl, 20 °C. These two alternatives can be tested experimentally. If the U(s) species are formed in the unfolding step already, then their concentration should be insensitive to changes in the refolding conditions. If, however, U(s) is formed early in refolding in a reaction that competes with the reactivation of U(f) then the amplitude of the slow reaction should increase when cyclophilin is included in the refolding solution in order to accelerate prolyl isomerization relative to the fast folding of U(f).

The reactivation kinetics of U(f) are indeed changed dramatically when 1 µM cyclophilin is added to the refolding solution (Fig. 3). Two effects are observed. First, the fast reactivation reaction has virtually disappeared, and second, the slow reactivation reaction is markely accelerated. This result excludes the first and strongly supports the second alternative. The fast reactivation of HCAII and one or more prolyl isomerizations in the molten globular intermediate appear to be similar in rate, and therefore compete with each other. Cyclophilin exerts a dual role in these experiments. Immediately after transfer of the U(f) molecules to the refolding conditions it accelerates the formation of molten globular intermediates with incorrect prolyl isomers. Later, in the slow reactivation of these U(s) molecules cyclophilin then catalyzes the reisomerization of the initially introduced incorrect isomers. The catalyzed slow folding reaction in Fig. 3can be fitted to a double exponential reaction with rate constants of k(2) = 1.2 times 10 s and k(3) = 2 times 10 s and fractional amplitudes of A(2) = 0.6 and A(3) = 0.2. It should be noted that these rates are much higher than those observed for long-term denatured HCAII in the presence of 1 µM cyclophilin (cf. Table 1).

Effect of Cyclophilin on the Refolding of Short-term Denatured HCAII

To further elucidate the effect of cyclophilin on the refolding of the U(f) molecules, the cyclophilin concentration in the reactivation buffer was varied from 0 to 5 µM. A striking result was obtained when the amount of protein reactivated after 30 s was plotted as a function of cyclophilin concentration (Fig. 4). It decreased dramatically from about 50% in the absence of cyclophilin to a minimum of about 20% near 1 µM cyclophilin and then increased again to about 37% at 5 µM cyclophilin. This profile confirms the dual role of cyclophilin in the folding of U(f) molecules. Obviously cyclophilin catalyzes the isomerizations that lead from U(f) to U(s) early in refolding with very high efficiency and therefore the extent of reactivation decreases strongly between 0 and 1 µM cyclophilin. Above about 1 µM cyclophilin almost all U(f) molecules are converted to U(s) and thus cannot refold rapidly. Prolyl isomerizations become rate-limiting for the reactivation of apparently all molecules and are catalyzed by cyclophilin as well. This explains why the extent of reactivation after 30 s increases again, when the concentration of cyclophilin is further increased.


Figure 4: Reactivation yield for short-term denatured HCAII in the presence of 0-5 µM cyclophilin after 30 s of reactivation (bullet). 17 µM HCAII was denatured in 5 M GdmHCl, 0.1 M Tris/SO(4), pH 7.5, for 10 s at 2 °C and reactivation started by rapid 17-fold dilution with 0.1 M Tris/SO(4), pH 7.5, at 20 °C containing 0-5 µM cyclophilin. The reactivation yield after 30 s was determined using the discontinuous assay method.



U(s) molecules that were produced from U(f) early in refolding at the stage of the molten globule differ strongly from U(s) molecules that were produced by a long-term incubation under denaturing conditions with respect to the catalysis of reactivation by cyclophilin (Fig. 5). For long-term denatured HCAII, catalysis is rather poor and a nonlinear dependence of the apparent rate constant k(2) on cyclophilin concentration is observed. In the refolding of U(s) molecules that were produced from U(f) during refolding, catalysis by cyclophilin is much more efficient and k(2) depends almost linearly on cyclophilin concentration. 5 µM cyclophilin increases k(2) of long-term unfolded U(s) 2.4-fold, but k(2) of U(s) which is produced from U(f) 30-fold.


Figure 5: Rate constants k(2) of the medium slow steps in refolding of long-term denatured (circle) and short-term denatured (bullet) HCAII as a function of the concentration of cyclophilin. Refolding at 20 °C was measured in 0.1 M Tris/SO(4), pH 7.5, with the continuous assay method. HCAII concentration was 1 µM in all experiments. Experimental data for reactivation were analyzed as the sum of two exponentials. Here the faster step is defined as the medium slow step.



Prolyl Isomerizations in Unfolded HCAII

To analyze the kinetics of the isomerizations that occur in unfolded HCAII and lead to the disappearance of the U(f) species, we also performed double-mixing experiments. In the first step the protein was kept under unfolding conditions of 5 M GdmHCl at 2 °C for a variable period of time. Then, in the second step, samples were transferred to refolding conditions of 0.3 M GdmHCl, 20 °C, and the amplitude of the resulting fast refolding reaction was determined. The decrease of this amplitude with increasing duration of unfolding is shown in Fig. 6. Complex kinetics were observed, which could be approximated by the sum of 2 first-order reactions with rate constants of k(1) = 1.1 times 10 s and k(2) = 8.3 times 10 s and amplitudes of A(1) = 0.26 and A(2) = 0.23.


Figure 6: Time course of the decrease of fast refolding HCAII molecules (U(f)) as a function of denaturation time (bullet). 17 µM HCAII was denatured in 5 M GdmHCl, 0.1 M Tris/SO(4), pH 7.5, at 2 °C for the indicated time, respectively, and reactivation was started by rapid 17-fold dilution with 0.1 M Tris/SO(4), pH 7.5. The kinetics of reactivation were followed at 20 °C using the discontinuous assay method. The amplitude of the U(f) molecules at reactivation time 0 was determined by extrapolation of the slow refolding reaction to 0 s. The solid line represents a fit according to two parallel first-order reactions. Reactivation was followed with the continuous assay method.




DISCUSSION

HCAII unfolds very rapidly in the presence of high concentrations of GdmHCl even at low temperature. Therefore two different collections of unfolded molecules (U(f) and U(s)) could be produced by varying the duration of unfolding. After a short 10-s unfolding pulse at 2 °C, HCAII is completely unfolded, but the native-like isomeric states of the prolyl peptide bonds are still retained, i.e. U(f) molecules with correct prolines are populated. After 1 h of unfolding at 20 °C, cis/trans-equilibria are reached at all 17 prolines in the unfolded protein, and a heterogeneous mixture of polypeptide chains with incorrect prolyl isomers, U(s), is formed.

The refolding of both U(f) and U(s) starts with the rapid formation (k(1) > 0.33 s) of a compact molten globule-like intermediate, which already displays a native-like secondary structure. Such an intermediate was also observed in the refolding of bovine carbonic anhydrase B(22, 23, 25, 26) . The formation of the molten globule is then followed for both U(f) and U(s) by rate-limiting folding reactions which control the regain of the enzymatic activity of HCAII.

The reactivation of the U(s) molecules is a slow and complex multi-exponential process. It is accelerated by cyclophilin, but the efficiency of catalysis is fairly poor. We suggest that all slow steps in the refolding of HCAII are limited in rate by prolyl isomerizations. The complexity of the process is not surprising, since HCAII contains 17 proline residues. A fraction of these prolines are presumably trapped in the interior of the protein by the formation of the molten globule and thus not readily accessible for cyclophilin.

The kinetics of reactivation of the U(f) molecules with native prolyl isomers are also complex. In addition to a fast reaction, slow, proline-limited steps were also observed. They do not originate from U(s) species that were already produced in the 10-s unfolding pulse, but they were formed after the transfer of the protein to refolding conditions at the stage of the molten-globule intermediate. Prolyl cis/trans-isomerizations continue to occur and compete with the rate-limiting steps of folding. Indeed, the reactivation of the U(f) molecules (k(1) = 7.3 times 10 s) and prolyl isomerization (10-10 s) show similar rates at 20 °C, and the fast reactivation reaction could be suppressed efficiently when the competing prolyl isomerizations were accelerated by cyclophilin.

Interestingly, the U(s) molecules that are produced by catalyzed prolyl isomerization early in refolding are different from the U(s) molecules that were produced by long-term unfolding. Their reactivation is slightly faster and is catalyzed with much higher efficiency by cyclophilin. When the U(s) molecules were produced by long-term unfolding, then 5 µM cyclophilin increased the major folding reaction about 2.4-fold. When the U(s) molecules were produced early in refolding, however, the major refolding reaction was increased about 30-fold.

This indicates that different sets of prolines isomerize under unfolding conditions and early in refolding in the presence of cyclophilin. The molten globule is formed very rapidly during refolding, and possibly only the exposed prolines are isomerized rapidly by cyclophilin in this partially folded intermediate. These prolines remain accessible in the course of further folding and, as a consequence, their isomerizations toward the native conformation are also well catalyzed. When U(s) is produced under unfolding conditions, some incorrect prolines are buried rapidly in the molten globule and thus not readily accessible for catalysis during refolding.

After unfolding the U(f) species decreases in a complex reaction as shown in the double jump experiments in Fig. 6. This is not surprising, regarding the high number of proline residues in HCAII. To a first approximation the U(f) species decrease in two reactions with apparent rate constants (k) of about 10 s and 10 s at 2 °C. Prolyl isomerizations of single prolines in oligopeptides and in unfolded proteins often show apparent rate constants of equilibration near 10 s at 0 °C(26, 27, 28, 29) . The faster reaction (k = 10 s) in unfolded HCAII could result from an anomalously rapid isomerization. Alternatively, it could be caused by the high number of trans-prolines in the protein, which, in the unfolded molecules, can isomerize independently of each other in parallel reactions. Thus U(f) would decrease with an overall rate that is equal to the sum of the individual isomerizations. Since the correct trans-state is strongly favored in the unfolded protein chains as well, the individual isomerizations have very small amplitudes and in total give rise to a reaction that comprises only a fraction of all unfolded molecules. In addition, two cis-prolines are present in native HCAII. One of them (Pro-202) was shown to be non-essential for refolding(16) , and therefore we speculate that the isomerization of the other cis-proline (Pro-30) could be involved in the formation of the U(s) species. The non-native trans-isomer of Pro-30 is strongly favored in the unfolded protein, and therefore U(f) further decreases in a slow reaction in the double jump experiments. The observed biphasic kinetics for the decrease of U(f) in Fig. 6can be reproduced well in a model calculation by assuming that 10 trans-prolines and a single cis-proline isomerize with rate constants of 10 s each to cis/trans-equilibria of about 0.1. These model calculations do, of course, not exclude other possible explanations for the results in Fig. 6. They indicate, however, that at least a part of the trans-prolines and one of the cis-prolines are important in the folding of HCAII. In addition to barnase(30) , iso-2-cytochrome c(15) , chymotrypsin inhibitor 2(31) , and procollagen(32) , HCAII is another candidate where cis trans-isomerization appears to be rate-limiting for refolding.

Several conclusions can be drawn with regard to the mechanism of folding of HCAII, the mechanism of folding in general and the function of cyclophilin as a catalyst of folding.

The unfolded state of HCAII is strongly heterogeneous and most if not all molecules contain non-native cis- and/or trans-prolyl isomers. In refolding these molecules can rapidly adopt the molten globule state, irrespective of the presence of incorrect prolyl peptide bonds. Some of them may be located in the interior, others at the surface of the molten globule. A molten globule with incorrect prolyl isomers was suggested to exist also for bovine carbonic anhydrase B(22, 23, 24) .

Most of the exposed and possibly also some of the buried prolines are located in flexible regions of the protein. Cyclophilin catalyzes efficiently the equilibration of these prolines in the molten globule state with native isomers, which is formed rapidly in the folding of U(f). Evidently the structure of the molten globule is not specific enough to stabilize, and thus select the native-like isomeric states. They are only fixed in the final slow steps of folding, which are thus coupled with and limited in rate by prolyl isomerization.

Generally, when the direct refolding reaction of U(f) species U(f) N and the isomerization of prolyl peptide bonds U(f) &lrhar2; U(s) proceed with similar rates, the two processes compete with each other and the prolyl cis/trans-equilibrium is in part re-established. This was also noted in the refolding of other proteins near the transition region and used to test the mechanism of folding(33) . This competition can be very efficient when multiple prolyl isomerizations lead away from U(f).

Cyclophilin acts as a classical enzyme in the catalysis of protein folding. In the folding of HCAII it catalyzes isomerizations of both cis- and trans-prolines, and the direction of the reaction is determined only by the stability of the isomeric state of each proline in the native protein. When the native prolines are not yet stabilized (as in the molten globule), then a rapid cis/trans-equilibration of accessible prolyl peptide bonds is catalyzed by cyclophilin. Intermediates of the molten globule type are substrates for cyclophilin. This may be relevant for de novo folding, because newly made polypeptide chains may collapse rapidly to a compact state similar to the molten globule early in cellular folding. The rapid formation of such ordered structure interferes, however, with the catalysis of isomerization of interior prolines. This is another indication that rapid formation of stable structure is not necessarily of advantage for efficient folding.


FOOTNOTES

*
This work was supported by the Deutsche Forschungsgemeinschaft (Fi 455/3-3), the Fond der Chemischen Industrie, and the Boehringer Ingelheim Stiftung. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 49-345-617294; Fax: 49-345-647126.

(^1)
The abbreviations used are: cyclophilin, recombinant human cytosolic cyclophilin with a molecular mass of about 18 kDa; HCAII, recombinant human carbonic anhydrase II; BCA, bovine carbonic anhydrase B; GdmHCl, guanidinium hydrochloride.

(^2)
To facilitate reading we used the expressions ``non-native prolines'' for the isomeric conformation of the prolyl peptide bonds, different from the conformation in the native structure and ``native prolines'' for the isomeric conformation of the prolyl peptide bonds as present in the native structure; ``U(f)'' for the HCAII molecules containing only native prolines and ``U(s)'' for HCAII molecules with prolyl peptide bonds in their thermodynamic cis/trans-equilibrium; ``proline limited folding'' for folding steps that are limited in rate by prolyl cis/trans-isomerization; ``short-term denatured'' HCAII for HCAII denatured in 5 M GdmHCl, 10 s at 2 °C and ``long-term denatured'' HCAII for HCAII denatured in 5 M GdmHCl, 1 h at 20 °C.


ACKNOWLEDGEMENTS

We thank Dr. Uno Carlsson, Linköping University, Sweden, for his interest and for providing recombinant human carbonic anhydrase II. Recombinant human cytosolic cyclophilin was generously provided by Dr. Kurt Lang at Boehringer Penzberg, Germany. The sequencing of the N terminus by Dr. Peter Rücknagel and determination of the molecular mass by Dr. Angelika Schierhorn is gratefully acknowledged.


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