(Received for publication, September 7, 1994; and in revised form, November 2, 1994)
From the
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) 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
) could be
produced. Surprisingly, only a fraction of U
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
.
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.
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 ()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. ()In the first experiments the protein was
initially unfolded for 1 h at 20 °C to produce U
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
molecules, which had not yet undergone prolyl
isomerizations. Their refolding was also measured and compared with the
folding of the U
molecules. Finally the duration of
unfolding in the first step was varied, and the kinetics of
interconversion between U
and U
in the unfolded
protein were followed by the decrease in the amplitude of the fast
reaction in the refolding step.
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.
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 () 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 H 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.
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) are produced by this brief unfolding pulse.
Again, after dilution to native solvent conditions, their refolding and
the influence of cyclophilin could be examined.
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, pH 7.5, for 1 h at 20
°C. Reactivation was started by rapid 17-fold dilution with 0.1 M Tris/SO
, pH 7.5, 20 °C in the absence
(
) and presence of 0.25 µM (
), 1 µM (
), 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.
Figure 3:
Reactivation kinetics of short-term
denatured HCAII in the absence () and presence (
) of 1
µM cyclophilin. 17 µM HCAII was denatured in
5 M GdmHCl, 0.1 M Tris/SO
, pH 7.5, for 10
s at 2 °C and reactivation started by rapid 17-fold dilution with
0.1 M Tris/SO
, 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 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
species are formed in the
unfolding step already, then their concentration should be insensitive
to changes in the refolding conditions. If, however, U
is
formed early in refolding in a reaction that competes with the
reactivation of U
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
.
The reactivation kinetics of U 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
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
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
= 1.2
10
s
and k
= 2
10
s
and
fractional amplitudes of A
= 0.6 and A
= 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).
Figure 4:
Reactivation yield for short-term
denatured HCAII in the presence of 0-5 µM cyclophilin after 30 s of reactivation (). 17 µM HCAII was denatured in 5 M GdmHCl, 0.1 M Tris/SO
, pH 7.5, for 10 s at 2 °C and reactivation
started by rapid 17-fold dilution with 0.1 M Tris/SO
, 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 molecules that were produced from U
early in refolding at the stage of the molten globule differ
strongly from U
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
on cyclophilin
concentration is observed. In the refolding of U
molecules
that were produced from U
during refolding, catalysis by
cyclophilin is much more efficient and k
depends
almost linearly on cyclophilin concentration. 5 µM cyclophilin increases k
of long-term unfolded
U
2.4-fold, but k
of U
which is produced from U
30-fold.
Figure 5:
Rate
constants k of the medium slow steps in refolding
of long-term denatured (
) and short-term denatured (
) HCAII
as a function of the concentration of cyclophilin. Refolding at 20
°C was measured in 0.1 M Tris/SO
, 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.
Figure 6:
Time course of the decrease of fast
refolding HCAII molecules (U) as a function of denaturation
time (
). 17 µM HCAII was denatured in 5 M GdmHCl, 0.1 M Tris/SO
, 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
, pH 7.5.
The kinetics of reactivation were followed at 20 °C using the
discontinuous assay method. The amplitude of the U
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.
HCAII unfolds very rapidly in the presence of high
concentrations of GdmHCl even at low temperature. Therefore two
different collections of unfolded molecules (U and
U
) 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
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
, is formed.
The
refolding of both U and U
starts with the rapid
formation (k
> 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
and U
by rate-limiting folding reactions which control the regain of
the enzymatic activity of HCAII.
The reactivation of the U 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 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
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
molecules (k
= 7.3
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 molecules that are produced by catalyzed prolyl isomerization
early in refolding are different from the U
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
molecules were produced by long-term unfolding,
then 5 µM cyclophilin increased the major folding reaction
about 2.4-fold. When the U
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 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 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
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
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
species. The non-native trans-isomer of Pro-30
is strongly favored in the unfolded protein, and therefore U
further decreases in a slow reaction in the double jump
experiments. The observed biphasic kinetics for the decrease of U
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.
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 species U
N and the isomerization of prolyl peptide bonds U
&lrhar2; U
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
.
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.