Originally published In Press as doi:10.1074/jbc.C000156200 on April 25, 2000
J. Biol. Chem., Vol. 275, Issue 25, 18619-18622, June 23, 2000
ACCELERATED PUBLICATION
Comparison of Folding Rates of Homologous Prokaryotic and
Eukaryotic Proteins*
Margit
Widmann and
Philipp
Christen
From the Biochemisches Institut der Universität Zürich,
CH-8057 Zürich, Switzerland
Received for publication, March 10, 2000, and in revised form, April 5, 2000
 |
ABSTRACT |
The rate of polypeptide chain elongation is up to
one order of magnitude faster in prokaryotic cells than in eukaryotes.
Here we report that the rates of in vitro refolding of
orthologous prokaryotic and eukaryotic proteins correlate with their
differential rates of biosynthesis. The mitochondrial and cytosolic
aspartate aminotransferases of chicken and aspartate
aminotransferase of Escherichia coli show pairwise sequence
identities of 41-48% and nearly identical three-dimensional
structures. Nevertheless, the prokaryotic enzyme refolded 6 times
faster (at 25 °C) than the eukaryotic isoenzymes after denaturation
in 6 M guanidine hydrochloride. Prokaryotic malate
dehydrogenase and lactate dehydrogenase also renatured faster than
their orthologous eukaryotic counterparts, suggesting that evolutionary
pressure has adapted the rate of folding to the rate of elongation of
polypeptide chains.
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INTRODUCTION |
The rate of polypeptide chain elongation in prokaryotes (1, 2) is
4-10 times faster than in eukaryotic cells (3, 4). Here, we address
the question of whether the faster rate of protein synthesis in
prokaryotes correlates with faster rates of protein folding. The
ultimate determinant of the folding rate of proteins is the primary
structure including proline content. However, other factors such as
protein size, chain topology, and thermodynamic stability are thought
to contribute to a wide range of kinetic patterns (5-7). For our
study, we chose three sets of homologous (or more precisely,
orthologous) eukaryotic and prokaryotic proteins (Table
I) to eliminate differences in size or
chain topology. The AspATs1
of chicken (mitochondrial and cytosolic isoenzymes) and of
Escherichia coli possess nearly identical structures (8).
All three enzyme variants are 
2 dimers, each subunit
composed of 13 -helices, a 7-stranded 
-sheet core, and one
molecule of the coenzyme PLP. The two other sets of orthologous
proteins that we examined were prokaryotic and eukaryotic MDH and LDH.
MDH is an 2-dimer with 8 -helices and 5-6
-strands/subunit, and LDH is a tetramer with 9 -helices and 6-10
-strands/subunit.
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Table I
Amino acid sequence identities of the investigated sets of orthologous
prokaryotic and eukaryotic proteins
The degree of identity was determined with the program SimAli (P. K. Mehta and J. Heringa, unpublished). In all cases, homology
has been confirmed by similarity in three-dimensional structure
(Protein Data Bank accession numbers: AspAT: 1asl, 2cst, 1tat; MDH:
1emd, 4mdh,1mld; LDH: 1ldb, 5ldh, 9ldt; the three-dimensional structure
of LDH from pig rather than chicken is known).
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EXPERIMENTAL PROCEDURES |
Proteins--
Mitochondrial and cytosolic AspAT were purified
from chicken heart as described previously (9). E. coli
AspAT was overproduced in strain TY103 harboring pKDHE 19/AspAT (kindly
provided by Dr. H. Kagamiyama, Osaka Medical College) and purified
according to published protocols (10). Prior to experimentation, all
AspATs were subjected to Sephadex G-25 gel filtration (Amersham
Pharmacia Biotech) to remove excess cofactor PLP. The concentrations of the purified enzymes in the PLP form were determined photometrically (
280 = 4.7×104
M
1 cm1
for E. coli AspAT and 280 = 7.0×104 M1
cm1 for both mitochondrial and cytosolic
AspATs; Ref. 9). Porcine mitochondrial, porcine cytosolic and E. coli MDH, as well as chicken muscle (type XXXIV), chicken heart
(type VIII), and Bacillus stearothermophilus LDH,
were purchased from Sigma.
Denaturation and Refolding--
Denaturation of AspAT was
verified by determining the number of sulfhydryl groups reactive with 1 mM 5,5'-dithio-bis(2-nitrobenzoic acid). For all proteins
tested, CD spectroscopy confirmed the complete loss of secondary
structure. Renaturation of AspAT was initiated by a 330-fold dilution
of the solution of the denatured enzyme in 6 M GdnHCl with
refolding buffer (25 mM Hepes-NaOH, 100 mM KCl,
1 mM 1,4-dithio-DL-threitol, pH 7.0) in
Minisorb tubes (Nunc). The concentrations of the proteins in the
refolding solution were 3 or 6 µg/ml, corresponding to subunit
concentrations of 67 and 134 nM if the refolding was
followed by enzymic activity or fluorescence, respectively. A
glass-coated magnetic stirrer was used in the former case, and manual
mixing was applied in the latter case. Refolding of MDH was initiated
by a 380-fold dilution to a final subunit concentration of 85 nM with refolding buffer (100 mM potassium
phosphate, 1 mM 1,4-dithio-DL-threitol, 1 mM EDTA, pH 7.4). Refolding of LDH was initiated by a
210-550-fold dilution to a final subunit concentration of 85 nM with refolding buffer (100 mM Tris-HCl, 1 mM 1,4-dithio-DL-threitol, 1 mM
EDTA, pH 7.3).
Measurement of Enzymic Activity--
A sample of the refolding
solution was transferred into the activity assay at the indicated
times. The ensuing dilution was 8-fold. The activity of the refolded
protein was calculated from the initial linear segment of the reaction
progress curve. AspAT activity was determined in a coupled assay with
1.8 units of MDH and 200 µM NADH in 50 mM
sodium phosphate, 20 mM aspartate, 20 mM
2-oxoglutarate, pH 7.5, at 25 °C. MDH activity was measured with
freshly dissolved 20 mM oxalacetate and 200 µM NADH in 100 mM potassium phosphate, pH
7.4, at 25 °C. LDH activity was measured with 30 mM
pyruvate and 200 µM NADH in 200 mM Tris HCl,
pH 7.3, at 37 °C. GdnHCl at the low concentrations present (1-4
mM) did not interfere with the activity measurements.
Fluorescence Measurements--
The intrinsic fluorescence of
AspAT (132 nM subunit concentration) was recorded in a
1 × 1-cm cuvette with a Spex Fluorolog spectrofluorimeter. For
kinetic refolding experiments, the wavelengths of excitation and
emission were set at 290 nm (bandwidths 1.8/1.8 nm) and 360 nm
(9.2/18.5 nm), respectively.
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RESULTS AND DISCUSSION |
The rates of refolding of the prokaryotic proteins after
denaturation in 6 M guanidine hydrochloride (GdnHCl) for 40 min proved without exception faster than those of their orthologous
eukaryotic counterparts (Fig. 1), the
ratios between the folding rates of the prokaryotic and eukaryotic
enzymes at 25 °C varying from 1.6 to 82 (Table
II). Lowering the temperature
increased the difference between the rates of folding of
prokaryotic and eukaryotic AspATs. At 2 °C, reactivation of
prokaryotic AspAT was 20 times faster than the refolding of the
eukaryotic enzyme; at 37 °C, the physiological temperature, the
prokaryotic enzyme still folded 5 times faster. The rate of
reactivation of AspAT (measured at 15 °C) was independent of protein
concentration in the range of 66 to 330 nM. Because only
dimeric AspAT is catalytically active (11), this finding indicates that
formation of the dimer was not rate-limiting. The thermodynamic
stability of the AspAT variants toward thermal and chemical
denaturation does not correlate with their rates of refolding. The
melting temperatures of E. coli and mitochondrial AspAT as determined by measurements of circular dichroism were 59 and 58 °C,
respectively, whereas that of cytosolic AspAT was 69 °C. The prokaryotic enzyme is more similar in stability to mitochondrial AspAT
than to the cytosolic enzyme also with respect to chemical denaturation.
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Fig. 1.
Reactivation of AspAT, MDH, and LDH. The
enzymes were denatured in 6 M GdnHCl, 50 mM
sodium phosphate, pH 7.5, at 25 °C for 40 min; renaturation was
initiated by dilution with refolding buffer at 25 °C (see
"Experimental Procedures"). The final protein subunit concentration
was 3 µg/ml corresponding to 66 and 85 nM for AspAT and
for MDH as well as LDH, respectively. For every set of orthologous
proteins, the same conditions were used (for details, see
"Experimental Procedures"). The addition of excess cofactor PLP
(0.1 mM) to the refolding buffer did not change the rate
and yield of reactivation of AspATs. Apparently, the intrinsic content
of cofactor sufficed to exclude PLP as a limiting factor.
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Table II
Comparison of the refolding rates of prokaryotic and eukaryotic
proteins
The rate constants (kobs for the first rapid phase)
were calculated from the data given in Fig. 1 and additional
experiments of the same kind.
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The difference in the rate of refolding was accompanied by a difference
in yield. In all cases tested, the prokaryotic proteins were obtained
at a significantly higher yield (Fig. 1). The yield of folding of
E. coli AspAT seems to be mainly under thermodynamic control
(5); the conformational isomers generated during refolding apparently
exist in rapid equilibrium, and the initial formation of incorrect
conformers is corrected by their conversion to the thermodynamically
most stable form. In contrast, the folding of cytosolic and
mitochondrial AspATs seems to be under kinetic control, i.e.
the rate of equilibration between intermediate conformational state is
slow compared with the rate of formation of the final conformational
state. Incorrectly folded conformers generated during renaturation
cannot reshuffle within a physiologically significant time interval.
Prokaryotic AspAT does not need the assistance of chaperones to refold
with high yield; however, the yield of mitochondrial AspAT, which
refolds spontaneously with very low yield, is markedly increased in the
presence of the Hsp60 (GroEL/GroES) and Hsp70 (DnaK/DnaJ/GrpE)
chaperone systems of E. coli (12).
The close structural similarity of the proteins compared within a given
set of homologs (Table I) allows the conclusion that the primary
structure rather than the length of the polypeptide chain or particular
features of the native spatial structure determines the specific
refolding behavior. All three AspATs possess two conserved
cis-peptidyl proline bonds (Pro-138 and Pro-195; the numbering corresponds to that of pig cytosolic AspAT) among a total of
15 to 20 proline residues (13-16). The two conserved cis proline residues lie in close spatial proximity to one another on the
surface of the large, coenzyme-binding domain. In E. coli AspAT, the two conserved cis proline residues have recently
been substituted with alanine. Neither mutation brought about
significant changes in three-dimensional structure, thermodynamic
stability, and enzymic activity (16). Apparently, the invariance of
these two cis peptidyl proline bonds is not due to
structural or functional reasons. The changes in folding rates of the
mutant enzymes suggest, however, that these cis proline
residues are located at positions within the polypeptide chain where
their isomerization might control the rate of folding (6, 17). Does
trans-cis proline isomerization underlie the observed
difference in refolding rates of prokaryotic and eukaryotic AspATs? To
approach this question, the dependence of the rate of refolding on the
duration of the denaturation period was examined. Refolding was
initiated after either 40-min or 40-s periods of denaturation at
25 °C. After the 40-s denaturation period, the cis
Xaa-Pro bonds may be assumed to have largely maintained their native
configuration (18). Their isomerization will thus not influence the
rate of refolding. After the 40-min denaturation, however,
trans isomers of proline peptide bonds may be expected to
predominate, the cis isomer content in short oligopeptides at equilibrium being only 10-30% (19, 20). Thus, if
trans-cis isomerization is slow, it might retard refolding.
By the criteria of both enzymic activity and intrinsic fluorescence,
E. coli AspAT refolded with the same rate when denatured for
either 40 min or 40 s (Fig. 2). In
contrast, the half-time of refolding of cytosolic and mitochondrial
AspAT decreased by a factor of 4 and 25, respectively, if the protein
had been kept in 6 M GdnHCl for only 40 s rather than
for the usual 40 min. The yield of refolding was in all cases the same
for short- and long-term denaturation. The enthalpies of activation for
refolding after a 40-min denaturation, as calculated from Arrhenius
plots (0, 15, 25, and 37 °C), were found to be 9, 16, and 20 kcal
mol1 for the activation of E. coli
AspAT, cytosolic AspAT, and mitochondrial AspAT, respectively.
Activation enthalpies in the range of 15-20 kcal
mol1 are generally thought to reflect Xaa-Pro
trans-cis isomerizations (19, 22-24). After a
40-s denaturation, the refolding rates (Fig. 2) and activation
enthalpies (~9 kcal mol1) of all three
AspATs were nearly identical. Apparently, Xaa-Pro isomerization is the
rate-limiting step in the refolding of cytosolic and mitochondrial
AspAT after long-term denaturation. This interpretation seems plausible
although both human cyclophilin A and peptidyl-proline cis-trans isomerase of E. coli, added in a
20-fold molar excess, failed to accelerate the reactivation of
cytosolic AspAT after a 40-min denaturation. Presumably, steric reasons
prevented the enzymes from catalyzing the trans-cis
isomerization. In E. coli AspAT, in which peptidyl-proline
isomerization is of course also an integral part of the folding
process, the isomerization is apparently facilitated by specific
effects of side chains adjacent in sequence or space (6, 25).
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Fig. 2.
Effect of duration of exposure to 6 M GdnHCl on the kinetics of refolding of AspATs. The
enzymes were denatured in 6 M GdnHCl for 40 min ( ,  )
or 40 s ( , - - -). The 40-s pulse in 6 M
GdnHCl at 25 °C was sufficient for complete denaturation, as
indicated by the CD spectra and measurements of catalytic activity. The
CD minimum at 220 nm of the native proteins disappeared completely in 6 M GdnHCl, with denaturation occurring within 20 s, the
dead time of handling. The temperature for refolding was 25 °C in
the case of E. coli and cytosolic AspAT and 15 °C in the
case of mitochondrial AspAT. Measurements with mitochondrial AspAT at
25 °C proved to be imprecise because of the low yield. The refolding
was followed by enzymic activity and intrinsic fluorescence.
Invariably, renaturation resulted in a blue shift of the emission
maximum and a decrease in intrinsic fluorescence intensity, which are
due to exposure of the tryptophan residues to a less polar environment
(21). The protein concentration was 66 and 132 nM for the
fluorescence and activity measurements, respectively. For details, see
"Experimental Procedures."
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CONCLUSION |
Are data on in vitro refolding relevant for in
vivo situations? In the cell, the folding of most proteins is
probably a cotranslational process or, in the case of certain proteins,
a cotranslocational process (26). However, the rates of the consecutive
steps in biosynthetic folding may be assumed to correlate with the
rates of the corresponding steps in the refolding of complete
polypeptide chains. The present data allow the following conclusions.
1) Homologous proteins with virtually superimposable three-dimensional
structures may markedly differ in their rate of folding. 2) Prokaryotic
representatives of a given protein fold faster than their orthologous
eukaryotic counterparts. Faster folding in prokaryotes in which chain
elongation is faster minimizes the occurrence of unfolded nascent
proteins. Conceivably, part of the difficulties encountered in
expressing eukaryotic proteins in prokaryotic cells might be due to a
combination of a fast rate of synthesis and a slow rate of folding,
which favors aggregation and proteolytic degradation. 3) In the case of
prokaryotic and eukaryotic AspATs, the rate of trans-cis
proline isomerization appears to have been modulated to control the
rate of folding, indicating yet another regulatory role (27, 28) of
peptidyl-proline isomerization.
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ACKNOWLEDGEMENT |
We thank Dr. William C. Merrick for helpful discussions.
| |
FOOTNOTES |
*
This work was supported in part by Swiss National Science
Foundation Grant 31-45940.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.
To whom correspondence should be addressed: Biochemisches
Institut, Universität Zürich, Winterthurerstr. 190, CH-8057
Zürich, Switzerland. Tel.: +41-1-635-5511; Fax: +41-1-635-5907;
E-mail: christen@biocfebs.unizh.ch.
Published, JBC Papers in Press, April 25, 2000, DOI 10.1074/jbc.C000156200
| |
ABBREVIATIONS |
The abbreviations used are:
AspAT, aspartate
aminotransferase;
PLP, pyridoxal 5'-phosphate;
MDH, malate
dehydrogenase;
LDH, lactate dehydrogenase;
GdnHCl, guanidine
hydrochloride.
| |
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Copyright © 2000 by the American Society for Biochemistry and Molecular Biology.
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ABSTRACT
INTRODUCTION
