Proline Isomerization Is Unlikely to Be the Cause of Slow Annealing and Reactivation during the Folding of Alkaline Phosphatase*

Eric DirnbachDagger §, Duncan G. Steel§parallel , and Ari Gafni§**Dagger Dagger

From the Dagger  Biophysics Research Division, ** Department of Biological Chemistry,  Department of Electrical Engineering and Computer Science, parallel  Department of Physics, § Institute of Gerontology, University of Michigan, Ann Arbor, Michigan 48109

    ABSTRACT
Top
Abstract
Introduction
References

The in vitro folding of Escherichia coli alkaline phosphatase (AP) from the guanidine hydrochloride (GdnHCl) denatured state is characterized by a significant slow phase in the post activational recovery of native protein lability (probed by the susceptibility to GdnHCl denaturation and occurring on the time scale of days) as well as a slow phase in the recovery of activity (on the time scale of minutes). Slow folding events have often been attributed to cis-trans isomerizations of X-Pro peptide bonds, a plausible explanation for AP, which contains 21 prolines per subunit. To investigate the role of proline isomerization in the two measures of refolding mentioned above, we have performed "double-jump" GdnHCl denaturation/renaturation experiments, with a third jump, where the rate of unfolding of refolded protein upon exposure to denaturant was added to assess the rate of change of lability. Our measurements of the time evolution of both the lability and the reactivation of refolded AP as a function of denaturation time show that proline isomerization is unlikely to be the cause of either of these slow events in the refolding of AP. The conclusions are further confirmed by the absence of proline isomerization effects when AP is refolded in the presence of human and periplasmic E. coli peptidyl-prolyl isomerase.

    INTRODUCTION
Top
Abstract
Introduction
References

Numerous studies of protein folding have shown that kinetic trapping can produce folded forms of the protein that differ from the native state and may in fact lead to long time scale changes in structure. The molecular origin of this behavior and the nature of the trapped state are unclear and of considerable interest in studies of folding. For example, in vitro refolded beta -lactoglobulin adopts a non-native conformation, as shown both by antibody binding (1) and by room temperature phosphorescence and lability (susceptibility to GdnHCl1 denaturation) studies (2). alpha -Lytic protease and subtilisin both require propeptides as intramolecular chaperones for successful folding, and the folding of either protein without the propeptide results in a stable molten globule structure that can convert to the native conformation only with the addition of the propeptide region in trans (3, 4). If subtilisin is folded with a mutated propeptide, it adopts a distinctly altered conformation (4). In these cases, the protein structure appears to be confined indefinitely in a near-native state. However, in other cases slow structural changes toward the native state have been observed. For example, in vivo newly synthesized phosphoglycerate kinase adopts a conformation that is slowly converted to a more stable state over time (5, 6). When the "old" (stable) form is unfolded in vitro, it adopts the "young" conformation upon initial refolding and then spontaneously recovers the old state (7, 8). The difference between stable, non-native state, kinetic trapping and slow conformational shifting toward the native state is likely due to the height of the activation energy barrier directly between the states, with a high barrier rendering the native state conformationally inaccessible on biologically relevant time scales.

Of particular interest to this paper is the recent report of structural "annealing" in alkaline phosphatase (AP) (9) where, as we discuss below, refolding from the extensively denatured state leads to unusually slow postactivational conformational changes reflecting a change in core rigidity and protein lability. This slow annealing behavior raises questions regarding the non-equivalence of the native and active states of AP. The native state represents the final pathway destination of the faster forming pre-annealed active-intermediate state or ensemble of states. The exact structural differences between the native and active states, and the molecular mechanism underlying these slow conformational changes remain unknown as does the relevance of this process to AP folding in vivo.

Slow events that take place during protein folding have often been attributed to cis-trans isomerization of X-Pro peptide bonds (10-13), an issue of particular relevance to AP, which is a homodimer with 21 prolines per monomer, all in the trans configuration (14-16). Upon unfolding of a proline-containing protein, the prolines equilibrate with a fraction of the denatured protein containing non-native cis/trans configurations. Refolding of the protein may then result in trapping of the non-native configurations which, depending on the activation energy for isomerization, may then evolve in time to the lower energy native state.

In the case of AP, measurements of the refolding kinetics show the existence of a slow phase in the recovery of lability, itself a measure of the activation energy to unfolding, on a time scale of days following the recovery of biological activity. This slow change in lability is accompanied by a slow change in the state of the core of the protein as inferred by a corresponding change in room temperature phosphorescence. Interestingly, the biological activity, which recovers relatively quickly, is also characterized by a slow phase on the order of minutes. Proline isomerization time scales within a folded protein are typically on the order of minutes (12) and hence are consistent with this slow time scale seen in reactivation. However, the unusually rigid core (9, 17) and large Delta G (18) for folding of AP raise the possibility that the time scale for isomerization of one or more X-Pro peptide bonds in the folded state could be unusually long. A misconfigured proline could possibly render the initially refolded AP more labile and would lead to the observed slow annealing.

To evaluate the significance of proline isomerization to the long time scale events in AP refolding, we performed a GdnHCl "double-jump" experiment, to study the effect of proline isomerization on reactivation (12), and a "triple-jump" experiment to determine the proline isomerization effect on lability. The basic experimental approach is shown in Fig. 1. In the first jump, AP is placed in a strong denaturant for varying lengths of time. Due to the high activation energy of approximately 20 kcal/mol for X-Pro isomerization (11), unfolding under strong denaturing conditions at low temperature uncouples the fast unfolding from the slow isomerization. The initially unfolded protein contains all prolines in their native (trans) configuration. However, with increasing incubation time in the denaturant, proline isomerization leads to an increasing accumulation of cis proline configurations in the AP chain. It is this increasing level of cis proline content that would lead to differences in refolding behavior, for these cis prolines must isomerize back to trans. In the second jump, the denaturant is rapidly diluted, refolding is initiated, and activity is measured as a function of refolding time for different denaturation times. The effect of proline isomerization on reactivation would then be detected by observing differences in the activity recovery rate. To examine the effect on lability, a third jump is added to the experiment where the refolded AP is placed in milder denaturing conditions, and the rate of unfolding is determined from the loss of activity. As in the case of reactivation, the effect of proline isomerization is detected by changes in lability as a function of time at high denaturant concentration.


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Fig. 1.   "Double/triple-jump" experiment scheme. Jump 1, native (N) AP is denatured within 1 min in 6.5 M GdnHCl, pH 2.3, at 0 °C. Jump 2, at various denaturation times, unfolded (U) AP is refolded at 25 °C, by 100 × dilution into refolding buffer, to produce reactivated (R) AP. Jump 3, at various refolding times, the reactivated-annealing (Ra) AP is unfolded in 4.5 M GdnHCl, pH 8.0, to assess the lability at that time.

A further test of a role for proline isomerization is to refold AP in the presence of the enzyme peptidyl-prolyl cis-trans isomerase (PPI), which has been found to catalyze the slow proline isomerizations in several proteins during folding (19-21). We have tested the effects of two similar forms of PPI on the reactivation and annealing of AP, the human cyclophilin, and the Escherichia coli periplasmic PPI, an enzyme that may naturally interact with AP in vivo. These enzymes share 34% sequence identity, a similar beta -sheet barrel structure (22), and a nearly identical kinetic efficiency toward model substrates (23).

    MATERIALS AND METHODS

Enzymes and Reagents-- Purified E. coli AP, human cyclophilin, chymotrypsin, and all reagents were obtained from Sigma. E. coli PPI was obtained as a generous gift of Dr. J. C. A. Bardwell.

Preparation of AP Stock Solution-- AP was dialyzed against 100 mM Tris, 0.1 mM ZnCl2, 0.1 mM MgCl2, pH 8.0, concentrated with a Centricon 30 to approximately 60 mg/ml, and stored at 4 °C until use. Enzyme concentrations were determined spectrophotometrically at 278 nm using A2780.1% = 0.72 (24).

AP Activity Assay-- AP activity was followed with the standard AP spectrophotometric assay (25). 10-µl refolding AP sample was added to 0.9 ml of AP assay mixture (1.0 M Tris, 1 mM p-nitrophenyl phosphate). The change in absorbance due to the hydrolysis of p-nitrophenyl phosphate at 25 °C was monitored for 30 s at 410 nm (Milton Roy Spectrophotometer).

AP Unfolding (Jump 1)-- AP stock was mixed with denaturing buffer (8 M GdnHCl, 0.2 M glycine, pH 2.0), equilibrated at 0 °C. Final concentrations were 10 mg/ml AP, 6.5 M GdnHCl, 0.15 M glycine, 17 mM Tris, 17 µM ZnCl2, 17 µM MgCl2, pH 2.3. A control sample was also prepared where stock AP was added to 100 mM Tris, pH 8.0, to give the same final concentration of enzyme.

AP Reactivation (Jump 2)-- AP, denatured as described above, was diluted 100-fold into refolding buffer (100 mM Tris, 0.1 mM ZnCl2, 0.1 mM MgCl2, pH 8.0) at 25 °C, yielding an AP concentration of 0.1 mg/ml. Enzymatic activity was assayed as a function of time for 2 h. A control sample was also prepared where control AP (from above) was diluted 100-fold into the same refolding buffer, to which 65 mM GdnHCl and 1.5 mM glycine were added.

Lability Determination (Jump 3)-- To determine AP lability, the rate of loss of enzymatic activity was determined in 4.5 M GdnHCl. Aliquots from the refolding AP solution were added at five different refolding times to a denaturing buffer (8 M GdnHCl, 100 mM Tris, pH 8.0) at 25 °C, to give final concentrations of 44 µg/ml AP, 4.5 M GdnHCl, 100 mM Tris, 44 µM ZnCl2, 44 µM MgCl2. The enzymatic activity was assayed as a function of time for 1 h. Control AP (three different samples) was also subjected to the same lability experiment.

Analysis of Lability Data-- The activity values were expressed as a percentage of the starting activity of the refolded AP at that time. The lability of native AP was similarly determined with the activity in denaturant expressed as a percentage of the control AP. All inactivation curves were well fit by two exponentials,
% <UP>activity</UP>=100<FENCE>C<SUB>1</SUB>e<SUP><UP>−</UP><FR><NU>t</NU><DE>&tgr;<SUB>1</SUB></DE></FR></SUP>+C<SUB>2</SUB>e<SUP><UP>−</UP><FR><NU>t</NU><DE>&tgr;<SUB>2</SUB></DE></FR></SUP></FENCE> (Eq. 1)
with tau 1,2 representing the unfolding time constants, and C1,2 the pre-exponential amplitudes. This fit corresponds to the assumption that the isomerization of any one proline leads to a more labile form of the protein and that while additional isomerized prolines might lead to additional lability terms, the relative concentration of these more labile forms is small, and the corresponding exponential terms would be difficult to resolve. The set of five lability curves (as a function of refolding time) for each refolded AP sample, along with the three control curves, were analyzed globally (Excel) using a least squares analysis with two common unfolding time constants. The pre-exponential amplitudes were varied individually, with their sum set to 1. The rate of decrease of the amplitude of the more labile fraction (smaller tau ) over refolding time determines the annealing rate.

Analysis of Refolding Data-- The refolding data was fit to two exponentials, with a short (approximately 20 s) initial time delay included to improve the fit. The reactivation equation used was,
<UP>Activity</UP>=A<SUB>∞</SUB>−<FENCE>C<SUB>1</SUB>e<SUP><UP>−</UP><FR><NU>t<UP>−</UP>t<SUB>0</SUB></NU><DE>&tgr;<SUB>1</SUB></DE></FR></SUP>+C<SUB>2</SUB>e<SUP><UP>−</UP><FR><NU>t<UP>−</UP>t<SUB>0</SUB></NU><DE>&tgr;<SUB>2</SUB></DE></FR></SUP></FENCE> (Eq. 2)
with Ainfinity representing the final activity, t0 the time delay, tau 1,2 the time constants, and C1,2 the pre-exponential amplitudes. All refolding curves were analyzed globally, with two common reactivation time constants and individual amplitudes that were varied as free parameters whose fractional sum was set to 1 and whose individual values were determined by optimizing the fit. Curves were also analyzed individually using two exponential fitting functions to test the validity of the assumptions of common decay times used in the global analysis. AP activity values were expressed as a fraction of the final yield, by dividing by Ainfinity , with C1/Ainfinity and C2/Ainfinity representing the fractional reactivation amplitudes.

Activity Assay of PPI-- Both human and E. coli PPI activities were tested using the coupled assay with chymotrypsin and the model substrate N-succinyl-Ala-Ala-Pro-Phe-nitroanilide (20, 26). An increase in absorbance at 390 nm occurs as chymotrypsin cleaves the trans peptide. Various levels of PPI were mixed with 10 µM chymotrypsin and 50 µM peptide in 100 mM Tris, pH 8.0, at 10°C and were monitored at 390 nm for 5 min. The peptide with the trans configuration (greater than 85%) is cleaved within the deadtime of several seconds, while the cis peptide is cleaved after the slow cis-trans isomerization. Our PPI samples had catalytic efficiency values: human PPI kcat/Km = 6.7 µM-1 s-1, and E. coli PPI kcat/Km = 11.4 µM-1 s-1, which are in good agreement with established values (20, 23, 27).

Experiments with PPI-- AP samples were denatured for 1 h at 25 °C and refolded in the presence of 0, 0.2, and 0.05 µM human PPI and 1, 0.2, and 0.05 µM E. coli PPI. Reactivation and annealing data were collected and analyzed as described above.

    RESULTS

To ensure high time resolution in the jump experiments, the initial denaturation of the protein (jump 1) needs to be rapid. Under the conditions used in this study, i.e. 6.5 M GdnHCl at pH 2.3 and T = 0 °C, the complete denaturation of AP, as monitored by circular dichroism at 222 nm, was found to occur in less that 1 min (data not shown). All experiments of the present study allowed unfolding for at least 1 min before initiating reactivation (jump 2) in order to ensure complete denaturation (no residual secondary structure). The low temperature was used to increase the proline isomerization time relative to the time scale for denaturation.

The effect of proline isomerization on the lability of refolded AP was monitored using the triple jump approach. Samples of AP were initially placed in strong denaturant (jump 1) for periods ranging from 1 min to 1 h. The samples were subsequently refolded for 2 h (jump 2) and then subjected to denaturation by 4.5 M GdnHCl (jump 3). Fig. 2 shows these inactivation curves for all refolded AP samples as well as for the control AP. The curves obtained for the refolded protein are nearly identical, exhibiting clear biphasic behavior, and are strikingly different from the curve of control AP. The different inactivation curves all contain a fast and slow time constant, with an average of 5.3 min and 105.2 min, respectively. The inactivation time constants for individual AP samples were determined by the global fit of all curves obtained for that sample over refolding time (see "Materials and Methods"). These two time constants represent two distinct protein populations, with relative amounts represented by the fractional amplitudes. The more labile component represents approximately 90% of the active protein for all refolded samples following 2 h of refolding and 40% for the control sample. In the inset to Fig. 2, we plot the fraction of the more labile component after 2 h of refolding as a function of denaturation time (jump 1). The data show that at this time of refolding, there is no difference in the refolded protein, as reflected in the lability studies, regardless of denaturation time.


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Fig. 2.   Lability curves of reactivated AP after 2-h refolding. Lability data were determined by exposure to 4.5 M GdnHCl. AP had been initially denatured during Jump 1 for the following times: open circle , 1 min; bullet , 2 min; , 6 min; black-square, 15 min; triangle , 30 min; black-triangle, 60 min; black-down-triangle , control. Activity data are expressed as a percentage of starting activity of that sample before denaturation. All curves are fit individually to two exponentials (Equation 1), which averaged 5.3 and 105.2 min. Inset, more labile fraction as a function of initial denaturation time.

All AP samples were then allowed to continue to refold for 1 week, during which time AP lability was monitored in order to determine the enzyme's annealing rate. Fig. 3A shows the time evolution of the inactivation curves for the AP sample that had been denatured for 1 min. The data demonstrate the structural annealing that occurs long after the recovery of activity as reported earlier (9). All curves were globally fit to two optimal inactivation time constants, as mentioned above. The fraction of the more labile species decreases over refolding time, and this behavior constitutes our measure of annealing. Fig. 3B shows the normalized fraction of the more labile AP as a function of refolding time for samples which have been denatured for different times. In all cases, the normalized fraction of the more labile protein decreases from approximately 0.9 to 0.5 in the course of one week. These fractions were normalized assuming an equilibrium level of 0.4 for the more labile component, the level observed for control AP. Fig. 3C shows the rate of loss of the more labile component as a function of denaturation time, obtained from the slope of the different data sets in Fig. 3B. The average annealing time is 290 h and is clearly independent of denaturation time.


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Fig. 3.   Annealing of reactivated AP. A, lability curves for AP that had been denatured for 1 min. The refolding time of each lability curve is: 2 h (open circle ), 22 h (bullet ), 69 h (), 129 h (black-square); 165 h triangle , control (black-down-triangle ). These curves were globally fit to two common time constants. B, fraction of normalized more labile amplitude over refolding time for all denatured AP samples. Data were calculated from the set of lability curves as shown in Fig. 3A, for each denatured sample: open circle , 1 min; bullet , 2 min; , 6 min; black-square, 15 min; triangle , 30 min; black-triangle, 60 min. The decrease in this phase is a measure of the slow annealing over time. The data were fit to a single exponential to determine annealing time. C, annealing times for all initial denaturation times, derived from the slopes of the curves in B. The average annealing time is 290 h.

The effect of proline isomerization on the kinetics of reactivation of AP, initiated by jump 2, was determined by following the reactivation kinetics for 2 h as a function of the time between jump 1 (denaturation) and jump 2. Fig. 4 presents the data obtained for AP samples that have been denatured for periods ranging from 1 min to 1 h. The reactivation curves have been normalized to the final activity value Ainfinity for each data set. After 2-h refolding, the reactivation was 80-95% complete. To account for folding events that precede the recovery of activity, such as the monomer collapse and dimerization (28, 29), an initial time lag of approximately 20 s was included, which increased the accuracy of the fit. This agrees well with the rates of monomer collapse as shown by fluorescence and circular dichroism (data not shown). Global analysis of the data assuming a biexponential recovery yielded fast and slow reactivation times of 3.3 min and 32.6 min, respectively, with corresponding amplitudes of 0.7 and 0.3. The inset to Fig. 4 shows a plot of the fraction of slow reactivation component as a function of denaturation time (jump 1). The data show that the length of the initial denaturation has no discernible effect on the reactivation kinetics.


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Fig. 4.   Reactivation of unfolded AP. Reactivation was started at the following denaturation times: 1 min (open circle ), 2 min (bullet ), 6 min (), 15 min (black-square), 30 min (triangle ), 60 min (black-triangle). All curves were normalized with AP activity assay data expressed as a fraction of the final activity and were globally fit to 2 exponentials (Equation 2). Globally fit time constants are 3.3 min and 32.6 min for the fast and slow phase respectively. Inset, slow amplitude from global fit as a function of initial denaturation time.

The reactivation and annealing experiments were repeated following the addition of 0.2 and 0.05 µM human PPI, and 1, 0.2, and 0.05 µM E. coli PPI. All annealing rates, as well as reactivation rates and phase amplitudes, did not differ from a control sample with no PPI added (data not shown).

    DISCUSSION

E. coli AP is a 94-kDa homodimeric nonspecific phosphomonoesterase, containing 2 Zn2+ and 1 Mg2+ per subunit, which are necessary for structure and catalysis (30-32). In our present study, we have observed the following three characteristics during the refolding of AP from the GdnHCl denatured state: (i) in freshly refolded AP, 90% of the protein is highly susceptible to denaturation in 4.5 M GdnHCl compared with 40% labile fraction in the control; (ii) a prolonged postactivation annealing process occurs with an average time scale of 290 h, where the AP gradually shifts to the less labile state; (iii) AP reactivation displays a distinctly biphasic kinetics with approximately 70% of the activity recovering with a rate of 0.3 min-1 and 30% with a rate of 0.031 min-1.

Proline isomerization, a slow reaction that has traditionally been proposed to explain long-lived folding events in proteins, is certainly a reasonable hypothesis for any or all of the above observations, and the goal of this study was to test this proposition. The above double-jump experiment describes an established approach, which relies on the fact that proline isomerization is a relatively slow process and that varying denaturation times allow the initiation of refolding from protein states that contain different numbers of non-native prolines.

For this analysis it is necessary to estimate the number of cis prolines expected per subunit over the various denaturation periods used in this study. Individual proline isomerization rates within a polypeptide chain can vary widely, depending on sequence, chain length, and on the presence of conformationally restrictive disulfide bonds (11, 33). The large number of prolines in AP (21 per subunit) further complicates this analysis. Much of the current knowledge about the rates of protein proline isomerization has been obtained from studies with bovine ribonuclease A, and we will use these data as an estimate for the isomerization rates for AP prolines. Schmid (12) found that at 0 °C the rate of conversion between the fast and slow folding forms (which differ in the configuration of the Tyr-92-Pro-93 peptide bond) was 0.0011 s-1 (900 s). Other double jump experiments at 10.5 °C have shown a cis-trans isomerization rate of 0.0055 s-1 (34). Calculating a value for 0 °C from the latter data by using a 20 kcal/mol activation energy (11) yields a lower isomerization rate constant of 0.0014 s-1 (709 s). Since this value results in an earlier accumulation of cis prolines, and thus provides a more conservative estimate for our analysis, we have used this for the characteristic time constant for cis-trans isomerization of prolines in AP at 0 °C.

To estimate the average number of cis prolines as a function of denaturation time, we note that based on the x-ray determined structure of AP, all 21 prolines are taken to be trans at the initiation of the denaturation, while 20%, i.e. 4.2, are assumed to be cis at equilibrium (11, 12). The isomerization equation used was,
[<UP>Cis</UP>]=4.2<FENCE>1−e<SUP><UP>−</UP><FR><NU>t</NU><DE>&tgr;</DE></FR></SUP></FENCE> (Eq. 3)
which follows immediately from the solution to the coupled first order rate equations and knowledge of the equilibrium (i.e. t right-arrow infinity ) value of [cis]. The results for different denaturation times are given in Table I, where, for example, the calculation predicts that the sample denatured for 1 min would have on average 0.34 cis prolines, while the sample denatured for 60 min would closely approach the equilibrium value of 4.2 cis prolines.

                              
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Table I
Average number of cis prolines per AP chain during denaturation time
Expected number of cis prolines during denaturation at 0 °C, using previously determined trans-cis isomerization rates, were calculated using Equation 3. The time constant tau  = 709 s was calculated from a double-jump experiment at 10.5 °C (34). We have assumed throughout that all prolines in the fully denatured chain are equally likely to isomerize at similar rates; however it is possible that the His-129-Pro-130 bond in AP may isomerize faster due to intramolecular acid catalysis (35).

Taking the most conservative approach to the analysis of the data, i.e. assuming that isomerization of any one proline in AP has a disruptive impact during refolding that will cause slow events, let us examine the two extremes of denaturation time, the 1- and 60-min samples. One would expect that following 1 min unfolding ~66% of the AP molecules in the sample would still be in the all-trans configuration and hence rapidly refold directly into the native state (becoming fully enzymatically active and displaying the lability pattern of control AP). The anticipated slow reactivation phase of ~34% would then be attributed to the misconfigured cis AP. This agrees well with the observed 30% slow reactivation phase (Fig. 4). However, the observed lability curve for the 1 min sample displays a 90% more labile phase, which is much larger and inconsistent with the reactivation slow phase, if this is due to proline isomerization. At 60 min unfolding, in contrast, one would expect the full impact of proline isomerization to be manifested both in the reactivation kinetics and in the lability, as every AP chain would contain misconfigured cis prolines (average of 4), and both the more labile and slow reactivation phases would be expected to approach 100%. Our results (Figs. 2 and 4) clearly show that this does not occur and that the denaturation time has no discernible effect on either process, thus ruling out any significant role for proline isomerization.

The above stated conclusion is further strengthened by considering the annealing rates, for which similar arguments can be made. If proline isomerization affects AP lability, then one would expect that a greater number of cis prolines (generated during longer incubation times) should lead to a noticeable increase in the annealing time, since more prolines must reisomerize back to trans to produce the native enzyme. This contrasts with our observation that the annealing times for all refolded AP samples are nearly constant (Fig. 3, B and C), with an average of 290 h and no increase in this time is observed with longer denaturation. The annealing times appear to be unaffected by cis content, and the proline hypothesis fails in this instance as well.

The experiments repeated in the presence of PPI confirm the above results. PPI has been shown to catalyze the isomerization of mis-configured X-Pro bonds in folding proteins (19-21). It provides a more direct test of proline isomerization as a rate-limiting step. However, in the case of refolding in the presence of PPI, a negative result as we have found here may imply that a proline was simply inaccessible to the PPI and hence does not conclusively rule out proline isomerizations. An example would be a deeply buried proline that would be difficult for the PPI to bind and isomerize. For this reason, PPI experiments, when showing no effect, would be inconclusive in themselves. Taken together with the double-jump experiments shown above, they provide strong evidence for the absence of proline isomerization as a rate-limiting step in the refolding of AP.

The experiments described above show that neither the extreme lability of freshly refolded AP, the slow annealing of the protein over time, or the slow reactivation phase are likely to be caused by the isomerization of one or more of the 21 X-Pro peptide bonds in AP. This conclusion was reached under the assumption that all prolines are "essential" (10) and will cause observable slow refolding effects when isomerized. A similar conclusion can be reached, given the experimental data, if one makes the arguably more realistic assumption of a lesser number of essential prolines. An analysis of the x-ray structure of AP shows that all but 3 prolines are surface-exposed to some extent. One would expect that the most buried of the prolines would be less tolerant of misconfiguration and thus most disruptive in the cis form. If this is the case, one would still expect refolding differences as a function of cis content, as more of the AP during the initial denaturation accumulate these few essential prolines.

It appears from the results of this study that there are no prolines in AP that become disruptive in the cis configuration. This is consistent with a computational analysis of proline configurations in bovine pancreatic trypsin inhibitor, where in many cases, the structure was able to accommodate non-native prolines with little destabilization of native structure (36). At equilibrium, we would expect approximately 4 cis prolines in denatured AP, equally distributed in all combinations among all 21 prolines. These cis-rich forms of AP have similar annealing and reactivation behavior as the chains with lower cis content. This surprising non-heterogeneity in refolding is similar to the results of studies on the 9 proline penicillinase (37) and the 8 proline fibronectin type III module (38), where the expected effects of proline isomerization were not found. It thus seems that all possible combinations of cis prolines in refolding AP may incorporate into the folded chain and isomerize gradually without affecting lability, annealing, or reactivation. Thus, since proline isomerization does not appear to represent any rate-limiting folding step, it remains unclear whether there were any proline interactions with PPI during folding.

In summary, the multi-jump and PPI experiments described above demonstrate that proline isomerization is an unlikely origin of the slow time scale events in the postactivational recovery of AP lability or of the slow phase observed in the regain of activity following denaturation, indicating that the origin of this behavior is due to more complex structural behavior. It is possible that the lability changes are similar to the long time scale events seen during metal rebinding to apoprotein, which are due to equilibration between metal binding sites (39). Indeed, changes in lability represent shifts in the activation energy for unfolding, and it is well known that zinc plays a critical role in stabilizing AP (30-32). Alternatively, it is known that refolding of the thermally denatured apoenzyme occurs on a long time scale (18), and it may be that the structural changes associated with this reaction are also important in refolding of the holoenzyme. Present work is focusing on studies of the time scales associated with various structural signatures of the apomonomer and metal binding.

    FOOTNOTES

* This work was supported in part by NIA, National Institutes of Health Grant AG09761, National Institutes of Health Grant 5T32GM08270-06, Multidisciplinary Research Training in Aging Fellowship T32AG00114, and the Office of Naval Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Dagger To whom correspondence should be addressed: Rm. 913, N. Ingalls Bldg., University of Michigan, Ann Arbor, MI 48109. Tel.: 734-936-2156; Fax: 734-936-2116; E-mail: arigafni{at}umich.edu.

    ABBREVIATIONS

The abbreviations used are: GdnHCl, guanidine hydrochloride; AP, alkaline phosphatase; PPI, peptidyl-prolyl cis-trans isomerase.

    REFERENCES
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Abstract
Introduction
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