Catalysis of protein folding by parvulin1

J. Mol. Biol. (1997) 273, 752±762

Catalysis of Protein Folding by Parvulin Christian Scholz1, Jens Rahfeld2, Gunter Fischer2 and Franz X. Schmid1* 1

Laboratorium fuÈr Biochemie UniversitaÈt Bayreuth D-95440 Bayreuth, Germany

2

Forschungsstelle ``Enzymologie der Proteinfaltung'' der Max-Planck-Gesellschaft Kurt-Mothes-Str. 3 D-06120 Halle/Saale, Germany

Recently a new family of prolyl isomerases was discovered, which is unrelated with the cyclophilins or the FK-506 binding proteins. Parvulin, the smallest member of this new family, is a protein with only 92 residues, but parvulin-like domains occur in several large proteins that are apparently involved in protein folding or activation processes. We show here that, in addition to its activity in assays with proline-containing tetrapeptides, parvulin catalyzes the proline-limited folding of a variant of ribonuclease T1 with a kcat/Km value of 30,000 Mÿ1 sÿ1. This value is much smaller than the kcat/Km value of 1.1  107 Mÿ1 sÿ1 determined for the parvulin-catalyzed prolyl isomerization in the tetrapeptide succinylAla-Leu-Pro-Phe-4-nitroanilide. Parvulin itself unfolds and refolds reversibly in a simple two-state reaction with a Gibbs free energy of stabilization of 28 kJ/mol at 10 C. Most of the unfolded parvulin molecules refold in a slow reaction that involves prolyl isomerization and is catalyzed by cyclophilin, another prolyl isomerase. Moreover, parvulin accelerates its own refolding in an autocatalytic fashion, and the rate of refolding increases tenfold when the concentration of parvulin is increased from 0.5 to 3.0 mM. Apparently, small single-domain prolyl isomerases catalyze prolyl isomerization much better in short peptides than in protein folding reactions, presumably because the prolyl bonds are less accessible in refolding protein chains. It is possible that the additional domains of the large prolyl isomerases improve the af®nity for protein substrates. # 1997 Academic Press Limited

*Corresponding author

Keywords: protein folding; folding enzyme; prolyl isomerase; parvulin; autocatalytic folding

Abbreviations used: RNase T1, ribonuclease T1; RCM(S54G, P55N)-RNase T1, a variant of RNase T1 with the substitutions Ser54 ! Gly and Pro55 ! Asn, in which the two disul®de bonds are reduced and the cysteine residues carboxymethylated; FKBP, FK 506 binding protein; N, native protein; U, unfolded protein; k, rate constant of a chemical reaction; A, amplitude of a kinetic phase. {To facilitate reading we use the terms cis proline and trans proline for residues that are preceded by a cis or a trans peptide bond, respectively, in the folded protein and ``native-like'' and ``incorrect, non-native'' to denote whether in an unfolded state a particular prolyl peptide bond shows the same conformation as in the native state or not. Further, we use the expression ``isomerization of ProX'' for the isomerization of the prolyl peptide bond preceding ProX. The folding reactions that involve Xaa-Pro isomerizations as ratelimiting steps are demoted ``proline-limited'' reactions; the (usually) rapid folding of the unfolded molecules which have the Xaa-Pro peptide bond in the same conformation as the native protein is denoted as the ``direct'' folding reaction. 0022±2836/97/430752±11 $25.00/0/mb971301

Introduction Unlike other peptide bonds, those preceding proline often occur in the cis conformation in folded proteins (Macarthur & Thornton, 1991; Stewart et al., 1990). cis Prolyl bonds are well-suited to induce tight turns in the protein backbone. The cis/trans isomerizations about these bonds (prolyl isomerizations{) are intrinsically slow processes in oligopeptides, as well as in proteins (Stein, 1993), and they frequently determine the overall rate of the folding of a protein (Brandts et al., 1975; Kim & Baldwin, 1982, 1990; Schmid, 1993; Schmid et al., 1993). The ®rst enzyme that was found to catalyze a prolyl isomerization in a tetrapeptide was isolated from porcine kidney (Fischer et al., 1984). According to its activity, it was classi®ed as a peptidyl prolyl cis/trans isomerase (prolyl isomerase). Later it was found that this enzyme also accelerated slow protein folding reactions that are limited in rate by prolyl isomerizations (Lang et al., 1987; Lin et al., 1988). # 1997 Academic Press Limited

753

Catalysis of Protein Folding by Parvulin

Prolyl isomerases are ubiquitous enzymes, which occur in virtually all organisms and in all subcellular compartments (Fischer, 1994; Galat & Metcalfe, 1995; Schreiber, 1991). Surprisingly, two families of prolyl isomerases are also receptors for the immunosuppressants cyclosporin A (CsA) and FK 506. Accordingly, these two families which are unrelated, are now called cyclophilins and FK 506 binding proteins (FKBPs), respectively (Fischer, 1994). The trigger factor of Escherichia coli, an ef®cient folding enzyme, which is associated with the ribosome has a prolyl isomerase domain with a weak homology to the FKBPs (Callebaut & Mornon, 1995; Hesterkamp et al., 1996; Scholz et al., 1997; Stoller et al., 1995; Valent et al., 1995). Recently, Rahfeld et al. (1994a,b) discovered a novel prolyl isomerase in E. coli which could neither be inhibited by CsA nor by FK 506. This protein consists of only 92 amino acids and has therefore been called ``parvulin'' (from the Latin word ``parvulus'', which stands for ``very small''). It shares no sequence homology with cyclophilins or FKBPs. Homologies were found, however, with domains of PrsA from Bacillus subtilis (Jacobs et al., 1993; Kontinen & Sarvas, 1993), SurA from E. coli (Eisenstark et al., 1992), PrtM form Lactococcus lactis (Haandrikman et al., 1989; Nissen Meyer et al., 1992), Ptf1/Ess1 from yeast (Hani et al., 1995), and human Pin1 (Lu et al., 1996). These proteins and their genes have been identi®ed in strongly different screening procedures, but they all seem involved in protein folding or activation processes. This is particularly clear for the SurA protein. It is located in the periplasm of E. coli and participates in early stages of the maturation of outer membrane proteins. It was suggested that mutations in surA cause protein misfolding in the periplasm (Lazar & Kolter, 1996; Missiakas et al., 1996; Rouviere & Gross, 1996) and lead to the activation of a pathway which signals periplasmic protein misfolding to the cytosol (Missiakas & Raina, 1997; Rouviere & Gross, 1996). Pin1 is essential for the G2/M transition in mitosis and seems to recognize its substrate in a phosphorylation-dependent manner (Ranganathan et al., 1997). Parvulin shows a high prolyl isomerase activity towards tetrapeptides in the protease-coupled assay. With a speci®city constant kcat/Km of 1.69  107 Mÿ1 sÿ1 (for the substrate Suc-Ala-LeuPro-Phe-4-nitoanilide) it approaches the most active cyclophilins known to date (Rahfeld et al., 1994b). Parvulin is much more active than the FKBPs, but similar to them it prefers hydrophobic residues, such as leucine and phenylalanine, in the position preceding the proline in tetrapeptides (Rahfeld et al., 1994a,b). Here we asked whether parvulin can catalyze slow protein folding reactions that are limited in rate by prolyl isomerization. In addition, we investigated the conformational stability of this very small enzyme and the kinetics of its folding. Parvulin itself contains ®ve prolyl peptide bonds, and

we show that it catalyzes its own refolding in an autocatalytic fashion.

Results Catalysis of proline-limited protein folding To measure the activity of parvulin as a catalyst of a proline-controlled protein folding reaction we used a variant of ribonuclease T1, (S54G/P55N)RNase T1, as a substrate protein. Native wild-type RNase T1 contains two cis proline residues, Pro39 and Pro55, and their isomerizations are the ratelimiting steps in the folding of RNase T1. (S54G/ P55N)-RNase T1 is a variant of RNase T1 which contains only a single cis proline residue (Pro39). The folding mechanism of this variant is particularly simple and well characterized in molecular detail (Kiefhaber et al., 1990). Both the oxidized and the reduced form of (S54G/P55N)-RNase T1 were used in our folding experiments. When the two disul®de bonds are intact, the protein is highly stable and a largely folded intermediate forms rapidly during its refolding. Thus the incorrect trans Tyr38-Pro39 prolyl bond is already partially shielded from the solvent before it isomerizes to the cis state in the ®nal step of folding. The disul®de-reduced and carboxymethylated form (RCM(S54G/P55N)-RNase T1) is only marginally stable, and partially folded intermediates no longer accumulate during refolding. Therefore the Pro39 bond remains well accessible for catalysis of its trans !cis isomerization. The RCM form of (S54G/P55N)-RNase T1 has proven to be an excellent substrate for a wide range of prolyl isomerases (Schmid et al., 1993). Of all unfolded RCM-(S54G/P55N)-RNase T1 molecules, 85% contain an incorrect trans Pro39 and fold in a monophasic and reversible reaction, which is controlled in its rate by the slow trans ! cis isomerization about this prolyl peptide bond. In the absence of a catalyst it shows a time constant of 570 seconds at 15 C (Figure 1(a)). Parvulin strongly catalyzes this refolding reaction, and an almost 20-fold increase in the folding rate is observed when 1 mM parvulin is added (Figure 1(a)). The relative rate of catalyzed folding increases linearly with parvulin concentration (Figure 1(b)), and a catalytic ef®ciency of kcat/ Km ˆ 30,000 Mÿ1 sÿ1 can be calculated from the data in Figure 1(b). This is comparable to the values observed for the human prolyl isomerases Cyp18 and FKBP12, which are 73,000 and 13,000 Mÿ1 sÿ1, respectively, under the same conditions. However, this kcat/Km value for the catalysis of proline-limited folding is much lower than that for prolyl isomerization in the tetrapeptide Suc-Ala-Leu-Pro-Phe-4-nitroanilide, which under the same conditions is 1.1  107 Mÿ1 sÿ1. The initial velocity of catalyzed refolding increases linearly with the concentration of the substrate protein in the accessible range of concentrations (Figure 2). This suggests that folding

754

Catalysis of Protein Folding by Parvulin

Figure 2. Initial velocity n0 of the catalyzed refolding of RCM-(S54G,P55N)-RNase T1 as a function of the concentration of RCM-(S54G,P55N)-RNase T1 in the presence of 0.50 mM parvulin. Refolding was measured at 15 C in 2.0 M NaCl, 0.1 M Tris HCl (pH 8.0). The initial folding rates were determined and analyzed as described in Materials and Methods. For comparison the strong curvature of the enzyme kinetics of the triggerfactor catalyzed folding of RCM-(S54G,P55N)-RNase T1 is shown (under the same conditions, but at a trigger factor concentration of only 0.010 mM). These data are taken from Scholz et al. (1997).

Figure 1. Refolding kinetics of RCM-(S54G,P55N)RNase T1 in the presence of increasing concentrations of parvulin at 15 C. (a) The kinetics of refolding of 0.7 mM RCM-(S54G,P55N)-RNase T1, as followed by the change in ¯uorescence at 320 nm, are shown in the presence of 0, 0.25, 0.5, 0.75, 1.0, and 1.5 mM parvulin (from the bottom to the top). (b) Dependence on parvulin concentration of the rate of slow folding. The ratios of the observed rate constants in the presence, k, and in the absence, k0, of parvulin are shown as a function of the parvulin concentration. A value of 30,000 Mÿ1 sÿ1 is obtained for kcat/Km from the slope of the line in (b) (k0 ˆ 1.75  10ÿ3 sÿ1). Refolding of RCM-(S54G,P55N)RNase T1, in 0.1 M Tris-HCl (pH 8.0), was initiated by a 40-fold dilution from 0 M NaCl to 2.9 M NaCl/0.2 M urea in the same buffer.

protein chains bind only weakly to parvulin and that the Km value for RCM-(S54G/P55N)-RNase T1 is probably much higher than 10 mM. Accordingly, a permanently unfolded protein, such as reduced and carboxymethylated a-lactalbumin, does not inhibit catalyzed folding. These data indicate that similar to Cyp18 and FKBP12 parvulin shows a very weak af®nity to protein substrates. This is in contrast to the trigger factor from E. coli which catalyzes the slow refolding of RCM-(S54G/P55N)RNase T1 with much higher ef®ciency, because its af®nity for unfolded proteins is very high. It is almost saturated with substrate protein at a concentration of about 3 mM, and the Km value is 0.6 mM for RCM-(S54G/P55N)-RNase T1 (see inset in Figure 2; Scholz et al., 1997; Stoller et al., 1995). In contrast to parvulin, cyclophilin 18 and FKBP12 the trigger factor is a large modular protein, and

the prolyl isomerase domain alone is not suf®cient for the high af®nity towards protein substrates and thus for the high activity as a folding catalyst (Scholz et al., 1997). Rather, sites that are located outside the prolyl isomerase domain are responsible for the tight interaction with protein substrates (Zarnt et al., 1997). It is not known whether the additional domains in the large homologs of parvulin (such as SurA, PrtM or Pin1) could serve a similar function and modulate the prolyl isomerase activity. Role of accessibility for catalyzed folding As outlined above, the oxidized form of (S54G/ P55N)-RNase T1 with the two disul®de bonds intact regains most of its secondary and tertiary structure early in refolding and reaches a nativelike conformation in less than a second, even when the Tyr38-Pro39 bond is still in the non-native trans conformation. As a consequence, this prolyl bond becomes partially buried and thus less accessible for the catalysis of isomerization. It was found previously that the slow folding of oxidized (S54G/ P55N)-RNase T1 is indeed poorly catalyzed by Cyp18 or by FKBP12 (Schmid et al., 1993) and not catalyzed at all by the trigger factor (C. Scholz, unpublished result). For parvulin the catalytic ef®ciency in this folding reaction is also reduced (Figure 3). The kcat/Km value as calculated from the data in Figure 3(b) is about 2200 Mÿ1 sÿ1, which is 15-fold lower than the value for the RCM form (cf. Figure 1(b)). As with the other small prolyl isomerases, parvulin is therefore able to accept partially buried prolyl bonds as substrates, albeit with a strongly reduced ef®ciency.

755

Catalysis of Protein Folding by Parvulin

Conformational stability of parvulin The thermodynamic stability of parvulin was determined from the denaturant-induced unfolding transition (Figure 4(a)). Parvulin does not contain Trp residues, and therefore the transition was monitored by the increase in protein ¯uorescence at 305 nm which re¯ects the exposure of the Tyr sidechains upon unfolding. Identical transitions were observed at two different parvulin concentrations (0.5 mM and 3.0 mM), which demonstrates that the stability of parvulin is independent of its concentration and con®rms that it is a monomeric protein. Also, two different probes of unfolding, tyrosine ¯uorescence and amide circular dichroism, gave the same transition (Figure 4(b)), which suggests

Figure 3. Refolding kinetics of oxidized (S54G,P55N)RNase T1 in the presence of increasing concentrations of parvulin at 15 C. (a) The kinetics of refolding of 0.7 mM (S54G,P55N)-RNase T1, as followed by the change in ¯uorescence at 320 nm, are shown (from the bottom to the top) in the presence of 0, 1.0, 2.0, 3.0, and 4.0 mM parvulin. (b) Dependence on parvulin concentration of the rate of the two slow phases, (&) k1 and ( & ) k2 in the refolding of (S54G,P55N)-RNase T1. The ratios of the observed rate constants in the presence, ki, and in the absence, k0, of parvulin are shown as a function of the parvulin concentration. A value of 2200 Mÿ1 sÿ1 is obtained for kcat/Km for the Pro39-limited folding reaction from the slope of the line for k1/k0 in (b) (k0 ˆ 1.0  103 sÿ1). Refolding of (S54G,P55N)RNase T1, in 0.1 M Tris-HCl (pH 8.0), was initiated by a 40-fold dilution from 8.0 M urea to 2.9 M NaCl/0.2 M urea in the same buffer.

The slow refolding of oxidized (S54G/P55N)RNase T1, as shown in Figure 3(a), consists of two phases, both of which are catalyzed by parvulin. The major very slow reaction is the trans ! cis isomerization of Tyr38-Pro39 and the minor faster phase re¯ects the cis ! trans isomerization of the Trp59-Pro60 bond (Kiefhaber et al., 1990). This reaction is observed only in the folding of oxidized (S54G/P55N)-RNase T1, where it is coupled with a change in the ¯uorescence of Trp59. It is silent in the folding of the RCM form. The isomerization at Pro60 is catalyzed better than the one at Pro39 (Figure 2(b)), because, unlike Pro39, Pro60 is largely accessible in the native protein and presumably also in the partially folded intermediate.

Figure 4. Urea-induced unfolding transition of parvulin. (a) The increase in ¯uorescence at 305 nm is shown as a function of urea concentration for 3.0 mM (*, right ordinate numbering) and for 0.5 mM (*, left ordinate numbering) parvulin in 0.1 M Tris-HCl (pH 8.0), 1 mM EDTA, at 10 C. (b) Comparison of the unfolding transitions as measured by the increase in ¯uorescence (*) and by the decrease in amide circular dichroism at 220 nm (*) under the same conditions. The protein concentrations were 3 mM and 5 mM in the ¯uorescence and circular dichroism experiments, respectively. The fraction of native protein is shown as a function of the urea concentration. The continuous line in (a) represents a ®t to the data obtained at 3.0 mM parvulin and the broken lines represent the baselines for the ¯uorescence of the native and the unfolded protein, respectively, as obtained from the ®t. The continuous line in (b) represents the best ®t of the transition detected by circular dichroism. All ®ts were performed by using the procedure of Santoro & Bolen (1988) for the analysis of two-state unfolding transitions.

756 that the unfolding of parvulin is a two-state process. The transition midpoint is at 3.7 M urea. Unfolding is reversible, and the native ¯uorescence and CD signal as well as the original prolyl isomerase activity are regained after an unfolding/refolding experiment. An analysis of the transitions in Figure 4 based on the two-state approximation (Pace, 1986; Santoro & Bolen, 1988) gives a Gibbs free energy of stabilization of 28 kJ/mol for parvulin at 0 M urea and 10 C. Folding kinetics of parvulin Parvulin is in the unfolded state above 5 M urea (cf. Figure 4(a)). Above 6 M urea, unfolding is so fast that the ¯uorescence value of the unfolded protein is reached within the time required for manual mixing (three seconds). Folding and unfolding are usually slowest near the transition midpoint, where folded and unfolded molecules are populated in comparable amounts at equilibrium. The kinetics of equilibration between native and unfolded molecules at 3.9 M urea starting from either the unfolded or the folded protein (Figure 5) reveal that almost the same ®nal ¯uorescence value is reached as expected for a reversible reaction. Both unfolding and refolding are complex processes and seem to be composed of a fast reaction, which occurs in the range of about ten seconds, and a slow reaction in the time range of several hundred seconds. The ®nal ¯uorescence value is approached more rapidly in the unfolding than in the refolding experiment. This seems surprising, because in a two-state folding transition the kinetics of equilibration should be the same for unfolding and refolding when measured under identical ®nal conditions. We will see later that the slow phase of the unfolding/refolding of parvulin is limited by prolyl isomerizations and is in¯uenced by autocatalysis. The autocatalytic effect is

Catalysis of Protein Folding by Parvulin

stronger in unfolding (where, at time zero, 100% of the molecules are native) than in refolding (where, at the beginning, all molecules are unfolded and thus inactive as potential catalysts). Refolding in the region of the native baseline (0 to 2.5 M urea, cf. Figure 4) is a complex reaction. A fraction of the change in ¯uorescence is complete within the dead time of mixing. Further changes occur in the time range of about 1000 seconds. These changes become faster when the protein concentration in the refolding experiment is increased, and they are not easily decomposed into a sum of exponential functions. We will show below that this complexity of refolding also arises from prolyl isomerizations which are accelerated in an autocatalytic fashion, most notably at high parvulin concentration. Double-mixing experiments to identify prolyl isomerizations in the folding of parvulin The complexity and the low rates suggest strongly that prolyl isomerizations are rate-limiting events in the refolding of parvulin. Usually, unfolded molecules with correct prolyl isomers fold fast (they are called UF molecules), whereas unfolded molecules with incorrect isomers refold slowly (the US molecules). Prolyl isomerizations are intrinsically slow reactions, and, depending on the ¯anking amino acids they proceed with time constants of 50 to 500 seconds at 10 C. They can be further decelerated by decreasing the temperature, because the activation energy is high (Brandts et al., 1975; Schmid & Baldwin, 1978; Schmid et al., 1993). Therefore at low temperature, the UF species can be produced transiently by a short unfolding pulse, which is suf®cient for conformational unfolding in the N ! UF reaction (equation (1)), but not for the subsequent prolyl isomerization in the UF „ US equilibration. The direct refolding of the UF molecules can then be studied after a second mixing which transfers the protein to refolding conditions. When the duration of the unfolding pulse is increased, the UF „ US equilibration can occur, and the refolding of both UF and US can be studied after the second mixing: fast

slow

ÿÿ * ÿÿÿ * N) ÿ ÿ UF ) ÿ US

Figure 5. Kinetic traces observed for unfolding (lower curve) and refolding (upper curve) of parvulin in the transition region. Unfolding was initiated by a 6.5-fold dilution of native protein (in buffer only), and refolding was initiated by a 6.5-fold dilution of unfolded protein (in 6.5 M urea) to identical ®nal conditions of 3.9 M urea. The folding kinetics were measured at 10 C at a ®nal parvulin concentration of 4 mM, in 0.1 M Tris-HCl, (pH 8.0), by the change in ¯uorescence at 305 nm.

…1†

In the ®rst step of the double-mixing experiments parvulin was unfolded in 7.0 M urea at 0 C for either eight seconds (to populate UF) or 1800 seconds (to establish the UF „ US equilibrium). In the second step, the unfolded protein solution was diluted to 3.5 M urea at 10 C, and the kinetics of refolding were followed by ¯uorescence. Under these conditions, both the fast and the slow phase of refolding can be followed after each manual mixing and the prolyl isomerase activity of native parvulin is low. Therefore, there was only a small risk that native parvulin molecules, which are formed by the fast folding of the UF molecules, might catalyze UF „ US equilibration early in

757

Catalysis of Protein Folding by Parvulin

Autocatalysis in the refolding of parvulin

Figure 6. Refolding kinetics of parvulin after short-term (&) and after long-term unfolding (*), as measured by double mixing experiments at 10 C. For short term unfolding parvulin was exposed to 7.0 M urea for eight seconds in the ®rst step, before refolding was initiated in the second mixing by a 6.14-fold dilution to 3.5 M urea. For long-term unfolding the exposition to unfolding conditions in the ®rst step was extended to 1800 seconds. In both experiments the ®nal concentration of parvulin was 4.0 mM. The buffer was 0.1 M Tris-HCl (pH 8.0). Refolding was followed by the decrease in ¯uorescence at 305 nm. After short-term unfolding the refolding kinetics are well represented by the sum of two exponential functions. The major (folding) reaction is described by a rate constant of about 0.25 sÿ1 (A ˆ ÿ4.72) followed by a small unfolding reaction with a rate constant of about 0.003 sÿ1 (A ˆ ‡0.76). Refolding after long-term unfolding can also be represented by the sum of two exponential functions with rate constants of 0.07 sÿ1 (A ˆ ÿ1.147) and 0.0021 sÿ1 (A ˆ ÿ3.415), as indicated by the continuous line.

refolding and thus complicate the analysis of the double-mixing experiments. The results are shown in Figure 6. The UF molecules, produced by the short eight second unfolding pulse, refold indeed rapidly with a time constant of about four seconds. This fast UF ! N folding reaction is followed by a minor slow unfolding reaction, which is caused by the slow establishment of the UF „ US equilibrium (cf. equation (1)). It is coupled with N „ UF and thus leads to a small net unfolding of N molecules, which is re¯ected in the small increase in the ¯uorescence signal after extended time (Figure 6). After 1800 seconds of unfolding, the UF „ US reaction has reached its equilibrium in the unfolded protein and now refolding in the second step is very slow (Figure 6). A minor fast reaction is followed by a complex refolding reaction, which slows a half time of about 150 seconds. The strong difference in the rate of folding after a short and a long unfolding pulse, as in Figure 6, demonstrates that indeed a slow UF „ US equilibrium occurs after the conformational unfolding of parvulin and that at equilibrium most of the molecules are in the US state. The simplest explanation is that the UF „ US equilibrium re¯ects the cis „ trans isomerization of one or more of the proline residues of parvulin. The high proportion of US molecules suggests that at least one of the ®ve proline residues of parvulin is cis in the native protein.

Parvulin is a prolyl isomerase, and its slow refolding is apparently limited in rate by prolyl isomerization. Therefore it should catalyze its own refolding. A salient feature of an autocatalytic reaction is that the product accelerates its own formation and thus the reaction rate is expected to increase with reactant concentration. For a prolinelimited folding reaction, autocatalysis should lead to an increase in folding rate with protein concentration until the rate of direct fast refolding (UF ! N) is approached. Therefore we varied the concentration of parvulin between 0.5 and 3.0 mM in refolding experiments performed in 1.0 M urea, 2.0 M NaCl at pH 8.0, 10 C. The ¯uorescence of parvulin is weak and decreases to only a small extent during refolding (cf. Figure 4(a)). In the presence of 2 M NaCl, the change in ¯uorescence upon refolding was slightly increased, and thus the range of parvulin concentrations in the folding experiments could be extended to a lower limit of 0.5 mM. The rate of refolding of parvulin, as measured in 2.0 M NaCl/1.0 M urea (10 C), increases indeed strongly with the protein concentration (Figure 7), and the half time of folding decreases from 70 seconds at 0.5 mM to seven seconds at 3.0 mM. Very similar results, albeit with a lower signal-to-noise ratio, were obtained in the absence of NaCl under otherwise identical conditions. The rate of the fast UF ! N reaction is not yet reached at 3.0 mM parvulin. Double-mixing experiments as in Figure 6 indicated that under the conditions of Figure 7 the UF ! N reaction is complete within the dead time of mixing. The refolding of parvulin is also strongly accelerated by another prolyl isomerase. When 0.5 mM human cyclophi-

Figure 7. Dependence on protein concentration of the refolding kinetics of parvulin. Refolding of (*) 0.5 mM, ( & ) 0.75 mM, and 3.0 mM (&) parvulin was followed by the decrease in ¯uorescence at 305 nm. (*) Refolding of 1.0 mM parvulin in the presence of 0.5 mM human cyclophilin 18. Refolding was initiated by diluting the unfolded protein (in 7.0 M urea, 0.1 M Tris-HCl (pH 8.0), 10 C) to 1.0 M urea/2.0 M NaCl in the same buffer, 10 C. For the comparison the relative changes in ¯uorescence are shown. The absolute amplitudes and the ®nal values of the kinetics increased linearly with protein concentration.

758 lin18 is present during refolding the half time of refolding is further reduced to about three seconds (Figure 7). Higher concentrations of cyclophilin 18 could not be used, because its strong Trp ¯uorescence provides a very high back ground signal for measuring the small changes in the Tyr ¯uorescence during the folding of parvulin. Concentration-dependent reactions as in Figure 7 are also observed in the refolding of oligomeric proteins when the association of subunits is ratelimiting for folding. In such a case, however, the protein stability increases with protein concentration, which is not observed for parvulin (cf. Figure 3(a)). Taken together, these results suggest that the folding of the small prolyl isomerase parvulin is itself limited in rate by prolyl isomerizations and that during refolding the native molecules accelerate the refolding of the still unfolded molecules in an autocatalytic fashion. Autocatalysis has also been found in the folding of FKBP12 (Scholz et al., 1996; Veeraraghavan et al., 1996).

Discussion Parvulin from E. coli is a very small, singledomain member of a new family of prolyl isomerases, which (unlike the cyclophilins and FKBPs) are not inhibited by immunosuppressants. In addition to its activity towards proline-containing tetrapeptides (Rahfeld et al., 1994a,b), parvulin also catalyzes proline-limited protein folding reactions and thus resembles the small prolyl isomerases cyclophilin 18 and FKBP12. The cellular function of parvulin is not yet known, but intriguingly, domains with a high homology to parvulin were found in diverse proteins, such as PrsA from B. subtilis (Jacobs et al., 1993; Kontinen & Sarvas, 1993), SurA from E. coli (Eisenstark et al., 1992), PrtM from Lactococcus lactis (Haandrikman et al., 1989; Nissen Meyer et al., 1992), Ptf/Ess1 from yeast (Hani et al., 1995), and human Pin1 (Lu et al., 1996; Ranganathan et al., 1997). Most of these proteins have been implicated in functions related with protein folding or with regulation. Parvulin does not only catalyze the refolding of the substrate protein RCM-(S54G/P55N)-RNase T1, but, similar to FKBP12 (Scholz et al., 1996; Veeraraghavan et al., 1996), it also accelerates its own refolding in an autocatalytic fashion. Most of the unfolded parvulin molecules refold in a slow reaction, which indicates that at least one of the ®ve prolines of parvulin is cis in the folded protein. The autocatalysis leads to a strong acceleration of refolding when the protein concentration is increased. Moreover, it leads to an apparent violation of a fundamental rule of chemical kinetics, which speci®es that under identical conditions (such as in the middle of an unfolding transition) the kinetics of equilibration should be identical, irrespective of the initial conditions. For an autocatalytic folding reaction, this rule does not apply.

Catalysis of Protein Folding by Parvulin

Unfolding is faster than refolding because the initial concentration of active folding catalyst is high in the experiment that starts from 100% native molecules, but essentially zero in the experiment that starts from 100% unfolded molecules. The three families of prolyl isomerases (parvulins, FKBPs, and cyclophilins) show no sequence homologies but share several properties. All families comprise small single-domain members (such as FKBP12, cyclophilin 18, and parvulin) as well as large members with several domains (such as FKBP52, trigger factor, cyclophilin 40, or SurA). The single-domain prolyl isomerases are all highly active towards accessible prolyl bonds in peptides with kcat/Km values up to 107 Mÿ1 sÿ1 (Fischer, 1994; Harrison & Stein, 1992; Rahfeld et al., 1994b). The catalytic activity in proline-limited protein folding reactions is much lower, with kcat/Km values that are often about 100-fold or more reduced relative to the values measured for peptide substrates (Fischer, 1994; Harrison & Stein, 1992; Rahfeld et al., 1994b; Schmid, 1993; SchoÈnbrunner et al., 1991). Two factors limit the activity in protein folding: the reduced accessibility of the prolyl bonds in the refolding protein chains and the low af®nity of the small prolyl isomerases for their substrates. It is possible that the additional domains in the large prolyl isomerases serve to improve the af®nity for protein substrates or to increase the selectivity for a certain subset of protein substrates. Indeed, in the trigger factor from E. coli, all three domains are necessary to create the exceptionally high af®nity of this protein for the folding protein chains (Scholz et al., 1997; Stoller et al., 1995; Zarnt et al., 1997). Corresponding data for large members of the parvulin family, such as SurA, are not yet available. The ubiquitous distribution and the existence of multiple forms suggest that prolyl isomerases serve many different functions in the cell. These functions are probably linked by the common requirement to recognize proline-containing segments of a protein chain, either in nascent proteins to facilitate their folding, or in folded proteins to facilitate conversions between alternative functional states of these proteins. Proline residues are commonly found in tight turns at the protein surface, and cis „ trans isomerizations can occur in folded molecules (Evans et al., 1987; KoÈrdel et al., 1990). They could thus be used as intrinsically slow conformational switches that can be regulated by prolyl isomerases. There is good evidence in vitro and in vivo that prolyl isomerases catalyze protein folding reactions (Matouschek et al., 1995; Rassow et al., 1995; Schmid, 1993; SchoÈnbrunner et al., 1991). Regulatory functions have been identi®ed as well (Wang et al., 1996), but in most cases it is not yet known whether the binding to proline-containing chain segments and/or the catalysis of their isomerization are of primary importance in these functions. The importance of binding to a proline-containing chain segment has clearly been established for the

759

Catalysis of Protein Folding by Parvulin

interaction between cyclophilin and the HIV gag protein (Franke et al., 1994; Gamble et al., 1996; Thali et al., 1994; Zhao et al., 1997). A comparison with the proteases might be helpful to rationalize the multiple and seemingly contradictory functions of prolyl isomerases. Proteases are also organized in several unrelated families and are found as domains of large specialized proteins. On the one hand, there are small proteases, which simply digest proteins and accordingly show a high catalytic activity and little substrate speci®city. On the other hand, there are highly specialized proteases, which bind selectively to a single substrate protein and cleave it with very high precision. Such proteases regulate important processes, such as blood clotting, ®brinolysis or complement ®xation. In addition to the actual protease domain these proteases usually contain regulatory domains which, for example, mediate the speci®c binding to the correct target proteins (for reviews, see Neurath, 1986a,b). We suggest that the catalysis of prolyl isomerization is used in a similarly broad fashion, either in plain protein folding or for regulatory reactions, which, unlike proteolysis, are readily reversible.

Materials and Methods Materials Parvulin and (S54G,P55N)-RNase T1 were puri®ed as described (MuÈcke & Schmid, 1994; Rahfeld et al., 1994a,b). The prolyl isomerase activity of parvulin was assayed by using a tetrapeptide substrate as described (Rahfeld et al., 1994b). (S54G,P55N)-RNase T1 was reduced and carboxymethylated by the procedure used for wild-type RNase T1 (MuÈcke & Schmid, 1994). The concentrations of oxidized and of RCM-(S54G,P55N)RNase T1 were determined spectrophotometrically (in a Kontron Uvikon 860 spectrophotometer) by using an absorption coef®cient of e278 ˆ 21,060 Mÿ1 cmÿ1 (Takahashi et al., 1970). For parvulin an e280 value of 2800 Mÿ1 cmÿ1 was calculated by using the procedure of Gill & von Hippel (1989). Urea (ultrapure) was from ICN Biochemicals. Catalysis of folding of (S54G,P55N)-RNase T1 (S54G,P55N)-RNase T1 (20 mM) with intact disul®de bonds was unfolded for one hour in 0.1 M Tris-HCl (pH 8.0), containing 8.0 M urea. Refolding at 15 C was initiated by a 40-fold dilution of the unfolded protein to ®nal conditions of 0.7 mM (S54G,P55N)-RNase T1 in 0.2 M urea, 2.9 M NaCl and the desired concentration of parvulin in the same buffer. The folding reaction was followed by the increase in protein ¯uorescence at 320 nm (10 nm band width) after excitation at 268 nm (1.5 nm band width) by using a Hitachi F4010 ¯uorescence spectrometer. The small contribution of parvulin to the ¯uorescence was subtracted from the measured values in the individual experiments. The slowest step of the observed refolding reaction is the trans ! cis isomerization of Pro39. Its rate constant was determined by using the program Gra®t 3.0 (Erithacus Software, Staines, UK). RCM-(S54G,P55N)-RNase T1 (28 mM) was unfolded by incubating the protein in 0.1 M Tris-HCl (pH 8.0), at

15 C for at least one hour. Refolding at 15 C was initiated by a 40-fold dilution of the unfolded protein to the same ®nal conditions as in the folding experiments with the oxidized protein. The folding reaction was also followed by the increase in protein ¯uorescence at 320 nm. The initial velocities of RCM-(S54G,P55N)-RNase T1 folding were determined from the progress curves of folding in the presence of 0.5 mM parvulin in 2.0 M NaCl, 0.1 M Tris-HCl (pH 8.0), at 15 C. Both uncatalyzed and catalyzed folding occur in these experiments. The relative contribution of uncatalyzed folding increases linearly with RCM-T1 concentration, and the initial rate of catalyzed folding would be progressively overestimated when determined simply from the initial slope of the progress curve of folding. Kofron et al. (1991) developed a method to account for both uncatalyzed and enzymecatalyzed prolyl isomerization in a peptide. This method was used to analyze the catalyzed folding of RCM(S54G,P55N)-RNase T1. The observed kinetics of folding in the presence of parvulin are described by the differential equation (2): d‰UŠ ‰UŠ ˆ ÿk0  ‰UŠ ÿ kcat  ‰parvulinŠ  dt ‰UŠ ‡ Km

…2†

In this equation d[U]/dt is the rate of folding of the unfolded protein U, ÿk0  [U] is the contribution of uncatalyzed folding, and ÿkcat  [parvulin]  [U]/ ([U] ‡ Km) is the contribution of catalyzed folding. kcat and Km are the catalytic rate constant and the Michaelis constant, respectively, and [parvulin] is the concentration of parvulin. A non-linear least-squares ®t of the observed folding kinetics to equation (2) was performed using the program EASY-FIT (Schittowski, 1993), and it was accounted for that only 85% of the unfolded RCM-T1 molecules contain an incorrect trans Pro39 (Mayr et al., 1996), i.e. [U]0 ˆ 0.85  [RCM-(S54G,P55N)-RNase T1]. The slow refolding reaction, which is analyzed here, originates from these molecules. The rate constant of uncatalyzed folding k0 was measured in reference experiments in the absence of parvulin at each concentration of RCM-(S54G,P55N)-RNase T1. Its value of k0 ˆ 0.00175 sÿ1, which was found to be independent of the concentration of RCM-T1, was used when the experimental data were ®tted to equation (2). The values for kcat and Km as obtained from this analysis were then used to calculate the initial rates of catalyzed folding v0 at the different substrate concentrations from equation (3): n0 ˆ kcat  ‰parvulinŠ 

‰UŠ0 ‰UŠ0 ‡ Km

…3†

The contribution ÿk0  [U] from uncatalyzed folding (equation (2)) increases linearly with the concentration of RCM-T1 and dominates the observed folding kinetics at high [RCM-T1]. Therefore, data at RCM-T1 concentrations higher than 10 mM were not used for this analysis. Equilibrium unfolding transitions of parvulin Native parvulin was incubated at ®nal concentrations of 0.5 or 3.0 mM in 0.1 M Tris-HCl (pH 8.0), containing 1 mM EDTA in the presence of varying concentrations of urea for 12 hours at 10 C. The extent of unfolding was determined for each solution by measuring the ¯uorescence emission at 305 nm (10 nm band width) after excitation at 280 nm (3 nm band width). The concen-

760 trations of the denaturant in the stock solutions and in every sample were determined by the refractive index (Pace, 1986). A non-linear least-squares ®t of the experimental data to the equation, given for a two-state transition (Santoro & Bolen, 1988), was used to obtain the transition midpoints. Folding kinetics of parvulin The kinetics of unfolding and refolding were monitored by the change in ¯uorescence at 305 nm (10 nm band width) after excitation at 280 nm (3 nm band width). The ¯uorescence changes were monitored continuously between 0 and 1080 seconds of unfolding or refolding and data points were taken every second. Afterwards, datapoints were taken every 60 seconds. For these points the ¯uorescence was measured for eight seconds and averaged. As a consequence, the kinetic traces in the Figures become smoothed after 1080 seconds of refolding. Between data collection, the excitation shutter remained closed to minimize radiation damage. All experiments were carried out in 0.1 M TrisHCl (pH 8.0) and varying concentrations of urea at 10 C. Refolding was initiated by a strong dilution (typically 40-fold) of the unfolded protein (in 7.0 M urea). To compare the folding kinetics at different protein concentrations, the observed changes in ¯uorescence were normalized by setting the total ¯uorescence change equal to 100. The ®nal ¯uorescence values reached after refolding and the measured amplitudes of refolding were both found to depend linearly on protein concentration. The folding mechanism of parvulin is not known, and in all cases the observed changes in ¯uorescence followed a complex time dependence. The contribution of slow isomerizations in the unfolded protein to the refolding kinetics was characterized by a double-mixing technique (Brandts et al., 1975; Schmid, 1986). In the ®rst mixing, native parvulin was unfolded by a 5.5-fold dilution with 8.6 M urea in 0.1 M Tris-HCl (pH 8.0), to give unfolding conditions of 24.5 mM parvulin in 7.0 M urea, 10 C. Refolding after a short time (eight seconds) or a long time (1800 seconds) of unfolding was then initiated by the second mixing, in which the unfolded protein was diluted 6.1-fold in the ¯uorimeter cell to give refolding conditions of 4.0 mM parvulin in 0.1 M Tris-HCl, 3.5 M urea (pH 8.0), 10 C. The resulting slow refolding reactions were followed by the decrease in ¯uorescence at 305 nm (excitation at 280 nm).

Acknowledgements We thank J. Balbach, M. Jacob, T. Schindler, V. Sieber, and S. Walter for stimulating discussions. This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.

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Edited by P. E. Wright (Received 12 May 1997; received in revised form 15 July 1997; accepted 15 July 1997)