(Received for publication, April 11, 1995; and in revised form, May 11, 1995)
From the
Results are presented on a peptide fragment (1013-1056)
from human DNA topoisomerase II
DNA topoisomerases II perform topological changes of DNA, such
as catenation/decatenation, knotting/unknotting, and modification of
the surpercoiling, by passing an intact double helix of DNA through a
transient double-stranded break made in a second helix of
DNA(1, 2) . These enzymes are implicated in DNA
replication, recombination, and DNA transcription and are essential in
mitosis and meiosis by intervening in condensation/decondensation and
segregation of chromosomes. They further exhibit structural roles by
probable associations to chromatin as they are a major component of the
nuclear matrix. Topoisomerases II also present a crucial interest in
the treatment of human cancers since they have been identified as the
preferential cellular target for number of clinically important
antitumoral drugs(3) .
The active form of topoisomerase II
has been proposed to be dimeric (4, 5, 6) ,
although the motif of dimerization is not identified. Recently,
Zwelling and Perry (7) have suggested on the basis of sequence
examination that a leucine-zipper (994-1021 segment) could be at
the origin of dimerization of human topoisomerase II
The coiling of
Here, we
tested the reality of a coiled-coil motif in human topoisomerase II
The method of Lupas et al.(30) was
then applied to the 1013-1056 sequence as well as to the other
Figure 1:
A, the two possible
coiled-coil structures (casesI and II) for
the segment 1013-1056 of human DNA topoisomerase II
Figure 2:
A polyacrylamide-SDS gel of the
1013-1056 human topoisomerase II
Figure 3:
A, dependence curves of the mean residue
molar ellipticity at 220 nm versus the 1013-1056 peptide
concentration (logarithmic scale) in water, pH 10 at: 5 °C
(
Figure 4:
A, The CD spectra of the 1013-1056
peptide recorded in water at pH 10 from 5 °C (bottomspectrum) to 70 °C (upperspectrum)
by 5° increments. The peptide was used at 50 µM concentration. B, the thermal melting profiles for
peptide concentrations of 16 µM (
The molar ellipticity at 220 nm of the
1013-1056 peptide, [
Figure 5:
GdnHCl (
Values of -6.8 and
-7.4 kcal/mol for the dimerization free energy,
The present study decisively indicates that the topoisomerase
II 1013-1056 fragment has the potential to adopt a native-like
structure favorable to dimer formation, without the participation of
the entire protein(40) . Actually, a similar conclusion has
been reached for thermolysin fragment 255-316 in its dimeric
state, which is essentially identical to that of the corresponding
chain region in the x-ray structure of
thermolysin(41, 42) . Moreover, the unfolding
thermodynamic parameters of this fragment dimer are similar to those
normally observed for small and globular proteins(43) . The
same proved true for chromogranin A and one of its constitutive
peptides from a comparative thermodynamic study of their
oligomerization properties (44) .
Our results show that the
human topoisomerase II
A striking feature of the present work consists of the free energy
Furthermore, our results do not allow retention of the 994-1021
segment, which partly overlaps the preferred 1013-1056 segment,
as a possible candidate to participate in the coiled-coil
(leucine-zipper) structure.
Our study thus provides an additional
example tending to prove that model studies on fragments are relevant
for the folding or interactions of intact
proteins(45, 46) . Based on a number of experimental
results, there is real hope that some deeper understanding of the
structural and energetic properties of protein domains can be gained by
looking closer the folding and self-association processes present in
isolated polypeptide fragments.
We thank J. P. Levillain and M. Le Maout for skilled
technical assistance in peptide synthesis and Profs. Ph. Laurent and D.
Bellet for valuable discussions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSIONS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
. This was selected using the
procedure of Lupas et al. (Lupas, A., Van Dyke, M., and Stock,
J.(1991) Science 252, 1162-1164) for its potential to
adopt a stable coiled-coil structure. The same theoretical treatment
rejected the segment 994-1021 proposed by Zwelling and Perry (Zwelling,
L. A., and Perry, W. M.(1989) Mol. Endocrinol. 3,
603-604) as a possible core for leucine-zipper formation. Our
experimental studies combine cross-linking and CD analysis.
Cross-linking establishes that the 1013-1056 fragment forms a
stable homodimer in solution. Effects of increasing peptide
concentration on CD spectra confirm that only the 1013-1056
fragment can undergo a coiled-coil stabilization from an isolated
-helix. Unfolding experiments further show that the coiled-coil is
more stable in guanidium chloride than in urea. Values of -6.8
and -7.4 kcal/mol for the dimerization free energy are determined
by thermal and urea unfolding, respectively. These are strikingly
similar to the value recently found for the dissociation/reassociation
of the entire yeast topoisomerase II from sedimentation equilibrium
experiments (Lamhasni, S., Larsen, A. K., Barray, M., Monnot, M.,
Delain, E., and Fermandjian, S.(1995) Biochemistry 34,
3632-3639), although their significance relatively to
topoisomerase II undoubtedly requires further analysis.
, as also
suggested by Caron and Wang(8) . This structure belongs to the
widespread coiled-coil motif initially characterized by Crick in 1953 (9) and consisting of several amphipathic
-helices wrapped
around each other in a slightly left-handed
supercoil(10, 11, 12, 13) . It is
omnipresent in the b-ZIP family (for basic
leucine-zipper)(14) , including DNA-binding proteins such as
transcriptional factors (15) and protooncoproteins/oncoproteins
as c-Fos and c-Jun, which all dimerize through a leucine-zipper
motif(16) . An extensively analyzed leucine-zipper is given by
the yeast transcriptional factor GCN4 (11, 12, 13, 17, 18) .
-helices optimizes the interchain packing of
apolar residues and also allows specific interchain ionic interactions
in proteins as varied as fibrous, globular, or membrane-spanning
molecules. The most striking feature of coiled-coil helices is the 4-3
or 3-4 hydrophobic repeat. This repeat, firstly identified by Hodges et al.(19) , consists of a repeating heptad sequence
designated by the letters a-g, in which the positions a and d usually present hydrophobic residues. The a and d residues fall on the same side of the helix and pack in a
regular ``knobs-into-holes'' pattern along the dimerization
interface(9, 11) . This creates a hydrophobic core,
which is critical for the dimerization process. Some charged residues
in e and g positions of adjacent heptads could stabilize
the dimer and contribute to the specificity of dimerization through
electrostatic interactions(20, 21) .
allowing its dimerization. Secondary structure predictions by the
Garnier Osguthorpe Robson (GOR) method (22, 23) as
well as circular dichroism experiments led us to discard the sequence
994-1021 proposed by Zwelling and Perry (7) and permitted
instead the identification of a 44-amino acid segment(1013-1056)
characterized by both a high
-helix potential and five 4-3
hydrophobic repeats typical of coiled-coil motives. Glutaraldehyde
cross-linking, detailed circular dichroism analysis, and derived
thermodynamic results confirmed the dimeric coiled-coil structure of
the 1013-1056 fragment in solution.
Peptide Synthesis and Purification
The
1013-1056 sequence peptide from human topoisomerase II
(NH
-T
VLDILRDLFELRLKYYGLRKEWLLGMLGAESAKLNNQARFILE
-COOH)
was prepared together with the 994-1021 fragment
(NH
-V
FKLQTSLTCNSMVLFDHVGCLKKYDTVLDILRDLF
-COOH)
according to solid phase synthesis on an Applied Biosystems model 431A
peptide synthesizer using Fmoc (9-fluorenylmethoxycarbonyl) chemistry
with 4-hydroxymethyl-phenoxymethyl-copolystyrene, 1% divinylbenzene
resin and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate activation. The peptides were cleaved from the
resin by a trifluoroacetic acid/1,2-ethanedithiol/thioanisole treatment
for 2 h at room temperature. The crude peptide was purified by high
performance liquid chromatography on a Beckman System Gold apparatus
using two different columns successively: a reverse-phase
(RP)(
)
C
Aquapore (22
1 cm;
20-µm particles; 330-Å porosity) with a linear gradient of
0-80% acetonitrile/water (with 0.1% trifluoroacetic acid);
followed by a RP C
Spherisorb (25
1 cm; 2-µm
particles; 300-Å porosity) with a linear gradient of 0-100%
acetonitrile/water (with 0.1% trifluoroacetic acid). In both cases, the
flow rate was 4 ml/min. Peptide purity was estimated higher than 90% by
amino acid analysis (6 N HCl; 1 h at 150 °C) on a Beckman
model 6300 amino acid analyzer, by analytical high performance liquid
chromatography using a RP C
Nucleosyl column (25
0.46 cm; 5-µm particles; 300-Å porosity) with a linear
gradient of 0-100% acetonitrile/water (with 0.1% trifluoroacetic
acid) with a flow rate of 1 ml/min and by Edman degradation on an
Applied Biosystems model 477A sequencing apparatus.
Glutaraldehyde Cross-linking
The glutaraldehyde
cross-linking reaction was carried out at 20 °C. The reaction
mixture contained 50 µM (260 µg/ml) of 1013-1056
peptide and 0.001% (v/v) glutaraldehyde. A control was achieved with
260 µg/ml lysozyme. This protein both behaves as a monomeric
molecule at any concentration and presents an appropriate molecular
weight (14,300) to follow the association intermediates of our
fragment. Aliquots of the reaction mixture (10 µl) were taken at
different times (0, 30, 60, 120, and 180 min) and were mixed with 2
µl of 1.5 M sodium borohydride. The reaction was stopped,
each time, by boiling after addition of 38 µl of sample buffer
(0.125 M Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 100 mM DTT (dithiothreitol), and 0.01% bromphenol blue). 16 µl of
cross-linking products were run on a sodium dodecyl sulfate-16.5%
polyacrylamide gel electrophoresis (SDS-PAGE) according to the protocol
of Schägger and von Jagow (24) . The gel resolution was
increased by adding a 10% spacing gel between stacking and separating
gels. Finally, the separated products were revealed by silver
staining(25) .
Circular Dichroism
Circular dichroism spectra were
recorded on a Jobin-Yvon Mark IV high sensitivity dichrograph linked to
a PC microprocessor. Optical cells with path lengths of 10, 1, and 0.1
mm were placed in a thermostable cell holder. All spectra were recorded
with a 1-nm step and were base-line corrected. Circular dichroism
measurements are reported as mean residue molar ellipticity
[]
(deg
cm
dmol
). The relative
content in
-helix was deduced according to Zhong and
Johnson(26) : %
-helix =
(-10), where
is the
circular dichroism per residue at 222 nm and is related to
[
]
by:
= [
]
/3300. The concentration
of the stock solution of 1013-1056 peptide was determined at pH
> 12, 20 °C, from UV absorption in the aromatic region. Such a
high basic medium was necessary in order to complete the ionization of
the two tyrosine residues present in the peptide; however, all the
dilutions were made at pH 10, that was enough to achieve dissolution.
Obviously, neutral pH would be preferred to conduct
association/reassociation experiments. However, inspection of our dimer
model does not suggest any significant change within the side-chain
groups participating in the coiled-coil stabilization between pH 7 and
pH 10. The peptide ellipticities were measured 1 h after dilution at
the chosen concentrations, these lying from 0.1 to 1200
µM, and 10 min after changes of temperature. These laps of
time allow to reach the new equilibrium. The stock solutions of
guanidine hydrochloride (GdnHCl) and urea used for the unfolding
experiments were 7.3 M and 8.8 M, respectively. These
concentrations were determined with refractive index
measurements(27) . For GdnHCl and urea unfolding experiments,
the samples were allowed to equilibrate in the presence of different
concentrations of denaturant for a minimum of 4 h at room temperature
before measuring the ellipticity.
Calculation of Thermodynamic Parameters from Thermal
Unfolding
Supposing a transition between two states (folded
dimer unfolded monomer), the molar fraction of dimer was
calculated from thermal unfolding curves as:
=
([
] -
[
]
)/([
]
-
[
]
) where [
]
and [
]
are the ellipticity at 220 nm
associated to monomer and dimer, respectively, and [
]
is the ellipticity at 220 nm at the considered temperature. The values
of [
]
and [
]
in
the transition region were obtained by extrapolating the pre- and
post-transition regions. The equilibrium constant, K, is
expressed as: K =
/2C(1 -
)
and, at T
, K
= 1/C [1], where C and T
are the peptide concentration and the
temperature at the half-transition, respectively(28) . For any
process at equilibrium, -RT lnK =
H
- T
S
. Equation 1 provides an
expression for K at T
in terms
of C. Plugging the equality given above, yields upon rearrangement:
1/T
= R lnC/
H
+
S
/
H
. This equation
corresponds to a straight line when the reciprocal of the melting
temperature (1/T
) is plotted against the
natural logarithm of the peptide concentration (lnC). The
slope and the intercept yield
H
and
S
, respectively.
Calculation of Thermodynamic Parameters from GdnHCl and
Urea Unfoldings
The denaturation curves were analyzed by
assuming that the transition between folding dimer/unfolding monomer is
a two-state event (F
2U). An
equilibrium constant, K
, can be thus obtained at
each individual GdnHCl and urea concentrations by: K
= 2 C (1 - f
)
/f
, where C is the peptide concentration and f
is the
folded fraction as determined from the ellipticity at 220 nm, namely: f
= ([
] -
[
]
)/([
]
-
[
]
) (27) . [
]
and [
]
are the ellipticities of the
unfolded and native states, respectively, and [
] is the
observed ellipticity at any particular denaturant (GdnHCl or urea)
concentration. The free energy of unfolding in the absence of
denaturant (
G
H
O) was estimated
by a linear extrapolation of the unfolding free energy at each
individual denaturant concentration to zero denaturant concentration,
according to:
G
=
G
H
O - m[denaturant], where m is the slope of the
straight line, d
G
/d[denaturant].
Identification of a Coiled-coil Motif in Human
Topoisomerase II
Originally, the purpose of this work was to experimentally
verify the reality of the leucine-zipper structure proposed on sequence
examination grounds by Zwelling and Perry (7) for the 994-1021
segment of the human topoisomerase II . In this aim, we estimated
the capacity of this sequence to adopt an
-helical structure using
the procedure of Biou et al.(23) based on the
combination of two prediction methods: one by homology and the other by
GOR III(22) . The results indicated that the 994-1021 segment
was unfavorable to helical structurization. Instead, we found that the
1013-1056 segment located in the minimal region necessary to the
activity of topoisomerase II (29) and which partially overlaps
the 994-1021 segment exhibited a high probability to be structured in
an
-helix.
-helical predicted segments of human topoisomerase II
in
order to evaluate their propensity for the coiled-coil structure. This
method is based on the relative frequency of occurrence of amino acids
at each position of the coiled-coil heptad repeat. The 1013-1056
segment presents the potentiality of having two possible 4-3
hydrophobic heptad repeats (noted as casesI and II in Fig. 1A). Almost all the residues found
in positions a and d are of hydrophobic nature. Thus,
they may form a hydrophobic interface that stabilizes the coiled-coil.
In comparison, even if in the 994-1021 segment proposed as a possible
leucine-zipper structure by Zwelling and Perry the leucine residues are
found in the desirable d position, more than half of the
residues in a position appear unfavorable to dimerization.
determined according to the Lupas et al.(31) procedure. The characteristic 4-3 hydrophobic repeats in a
and d positions are indicated. Figure also shows an alignment of
several type II DNA topoisomerase sequences: HumA, HumB, Dm, and Sc, namely Homo
sapiens (isoforms
(1013-1056) and
(1031-1056)), Drosophila melanogaster(9) ,
and Saccharomyces cerevisiae(3) , respectively. B, helical wheel representation of the coiled-coil (caseII) formed by the segment
1013-1056.
It
has been established that the ratio of charged to apolar residues is
indicative of the shape of protein containing a
coiled-coil(31) . In the 1013-1056 segment, the positions b, c, e, f, and g are not really
hydrophilic and the ratio of charged to apolar residues is found equal
to 0.6. This could account for a coiled-coil integrated in a globular
protein, which appears to be the case of topoisomerase II(32) ,
as it has been shown that the apolar groups can be more easily
accommodated in the core of a globular unit than in a cylindrical one.
Finally, case II, shown under the wheel representation in Fig. 1B, appears more appealing than case I as it
displays a conservation of residues at a and d positions
in DNA topoisomerases II of several species as diverse as the human and
the yeast ones (Fig. 1A).
Glutaraldehyde Cross-linking
Fast fixation of protein association intermediates has been
applied with success because of the accuracy and sensitivity of the
method (reviewed in (33) ). We used the 1013-1056 peptide
at 50 µM concentration, at which the peptide was shown to
adopt a coiled-coil structure by circular dichroism (see below). The
existence of cross-linked species was determined by SDS-PAGE (Fig. 2). Formation of a significant amount of dimer was
detected immediately after mixing the peptide with glutaraldehyde, and
the dimer remained the major oligomeric species from 0 to 3 h. After 3
h, we also detected small amounts of trimer, tetramer, and pentamer.
peptide submitted to
glutaraldehyde cross-linking (see ``Materials and Methods'').
The products were revealed by silver staining analysis. The positions
of molecular size standards (kilodaltons) are shown on the left, and those of the predicted oligomers formed by the
1013-1056 peptide are indicated on the right. T and MW, respectively, correspond to a control without
cross-linker and to the molecular size
standards.
The present cross-linking results can be considered as significant
for the following reasons. First, low concentrations of cross-linker
and peptide prevented nonspecific reactions. Second, no complex larger
than the pentamer was detected even after 3 h, with a relative amount
remaining insignificant compared to cross-linking of the dimer. This
accounts for the fact that the dimer is the most stable complex and
that formation of higher order oligomers is barely due to interactions
of the dimer with peptide molecules in its surrounding. Third, no
cross-linking of the monomeric molecule lysozyme used as control was
detected under similar conditions. Thus, the glutaraldehyde
cross-linking and subsequent SDS-PAGE experiments confirmed the
autoassociative properties of the 1013-1056 peptide and pointed
out the predominance of dimer over the largest oligomeric species.
Circular Dichroism Spectroscopy
The ability of the 1013-1056 peptide to form a
two-stranded -helical coiled-coil was monitored by circular
dichroism. Two criteria were used in the analysis: the helical content
and the degree of coiling. The first was calculated from the intensity
of the molar ellipticity at 220 nm, while the second was estimated from
the ratio between the intensities of the bands at 220 and 208 nm. The
ratio [
]
/[
]
is about 0.8 for a single-stranded
-helix and about 1.0 for
a two-stranded
-helical coiled-coil(34) . Changes of this
ratio have been explained as follows. The amide n
*
transition (220-nm CD band) is essentially responsive to the
-helical content(26) . On the other hand, the amide
* excitation band at 208 nm polarizes parallel to the helix
axis and is sensitive to whether the
-helix is single-stranded or
an interacting helix as in the case of the two-stranded coiled-coil.
The decrease in the parallel band intensity, together, in fact, with a
red shift of its maximum(35) , must reflect the conversion of a
rigid single-stranded
-helix to an
-helical coiled-coil
structure. Furthermore, clear-cut thermodynamic data reflecting the
dissociation/reassociation may be obtained from CD experiments using as
variables polypeptide concentration, temperature, or chemical
denaturants.
Peptide Concentration Effects
The 1013-1056
peptide CD spectra show two minima at 208-210 and 222 nm
indicative of a significant proportion of the peptide residues in the
-helical conformation(36) . The molar ellipticity value at
220 nm, [
]
, became more negative with
increasing peptide concentration (Fig. 3A). The
concentration value at the half-transition increased with the
temperature (5, 9, and 24 µM peptide at 6, 20, and 40
°C, respectively). An isolated
-helix appears marginally
stable in aqueous solution, and its stabilization requires interactions
provided by tertiary and quaternary structures. Thus, the
[
]
concentration dependence shown by the
peptide results from the stabilization of the
-helix through its
oligomerization. As the concentration of peptide is increased, the
monomer-oligomer equilibrium is shifted toward the formation of
oligomer which increases the
-helical content. At 200 µM peptide concentration, CD spectral analysis with the method of
Zhong et al.(28) provides
50% of
-helix
content. However, above 200 µM, the ellipticity at 220 nm
becomes concentration-independent, suggesting the complete association
of the fragment. We may note that, in the same conditions, the CD
spectra of the 994-1021 fragment reflected an
-helix content of
approximately 15%, which does not vary with the peptide concentration
between 166 µM and 5.2 mM. This confirms the weak
potential for the corresponding segment in topoisomerase II to adopt a
stable coiled-coil structure.
), 20 °C (
), and 40 °C (
). B,
dependence curve of
[
]
/[
]
versus the 1013-1056 peptide concentration in
water at pH 10, 5 °C.
Certainly more revealing for the
self-association of the 1013-1056 peptide in solution, the ratio
[]
/[
]
in CD
spectra at 6 °C was found to increase from about 0.85 at low
peptide concentration (<2 µM) to approximately 1.08 at
higher peptide concentration (>10 µM) (Fig. 3B). Moreover, the peptide concentration
corresponding to the half-transition was found to be the same (6
µM), whether the transition was monitored from the
ellipticity at 220 nm or the ratio
[
]
/[
]
. We
also observed the expected red shift in the maximum of the parallel
band (208 to 210 nm), which, when added to the other effects, accounts
for the stabilization of a coiled-coil structure from an isolated
-helix.
Thermal Unfolding
Occurrence of an isodichroic
point at 203 nm during the thermal denaturation of the 1013-1056
peptide (Fig. 4A) provides support for the existence of
a predominant two-state structural transition, which according to the
above results is presumed to correspond to the conversion of an
-helical coiled-coil into an unfolded monomer.
[
]
decreased with increasing temperature,
but the phenomenon was found quantitatively reversible with the
decrease of temperature. The thermal curves at different peptide
concentrations showed discernible cooperative transitions. A clear
dependence of the melting temperature, T
,
on peptide concentration could be detected (Fig. 4B)
with T
values of 33, 53, 67, and 77
°C for peptide concentrations of 16, 50, 100, and 160
µM, respectively. This concentration dependence of T
also characterizes an oligomeric
structure. The thermodynamic parameters were determined from the
thermal unfolding curves according to the procedure described under
``Materials and Methods'' (Fig. 4B, inset). The relative magnitude of
H
(-11.3 kcal/mol) and T
S
(-4.5 kcal/mol) indicate that the coiled-coil formation is
enthalpically driven. The free energy
G
was
found equal to -6.8 kcal/mol. This free energy is similar to
those values determined for
-helical coiled-coil models (37) and allows assessment of the stability of the coiled-coil
formed by the 1013-1056 peptide.
), 50
µM ([
), 100 µM (
), and 160
µM (
). The denatured fraction, f
, was calculated as f
= 1 -
, where
is the molar fraction of
dimer (see ``Materials and Methods''). Inset, the
plots of 1/T against the natural logarithm of the peptide
1013-1056 concentration yield the
H
and
S
values (
H
= -11.2 kcal/mol and
S
= -15 cal/mol
K).
Trifluoroethanol Effects
TFE can increase the
helicity of single-stranded peptides(37) . However, TFE is also
a denaturant of tertiary and quaternary structure stabilized by
hydrophobic interactions. For instance, it has been shown previously
that 50% TFE is enough to disrupt the two-stranded -helical
coiled-coil(34) .
]
, steadily
increased of 45% during the solvent change from plain H
O to
50% TFE, reflecting the induction of helical conformation by TFE (data
not shown). Above 50% TFE, the ellipticity,
[
]
, no longer increased. The ratio
[
]
/[
]
decreased from a value above 1 at 0-20% TFE to about 0.95
at 30%. Accordingly, the
-helical coiled-coil structure appears
to be stable up to 20% TFE and, above this percentage, disrupted into
isolated
-helices that may recover their standard parameters.
Guanidine Hydrochloride and Urea Unfoldings
The
GdnHCl and urea unfoldings allowed us to estimate the stability of the
coiled-coil formed by the 1013-1056 peptide by considering the
transition midpoints. [GdnHCl] and
[urea]
values at which 50% of the peptide is
unfolded were found equal to 2.2 M and 1.9 M,
respectively (Fig. 5). The free energy,
G
H
O, derived from the unfolding
curve, was 17.2 kcal/mol for GdnHCl and 7.4 kcal/mol for urea (Fig. 5, inset). We note the good agreement for
unfolding between the free energy determined from the thermal (6.8
kcal/mol) and that from urea (7.4 kcal/mol). On the other hand, GdnHCl,
which is a charged molecule, could potentially mask electrostatic
interactions and increase hydrophobic interactions but would become a
denaturant at higher concentrations(39) . Thus, the results of
GdnHCl unfolding might traduce the contribution of hydrophobic forces
to the coiled-coil stability. On the other hand, the urea is uncharged
and the stability values of urea unfolding would in principle reflect
the contribution of both hydrophobic and electrostatic interactions to
the stability of the coiled-coil.
) and urea (
)
denaturation profiles for the 1013-1056 peptide coiled-coil in
water at pH 10, 20 °C. Inset, the linear dependence of
G
versus GdnHCl (
) and urea (
)
concentrations allows a simple determination of
G
H
O
Dimer Stability
While a comparative study on the effects of GdnHCl and urea
is always rather difficult, the stability values obtained from urea and
temperature unfolding should be consistent, which is the case in the
present study. Here, both the transition midpoints and the free
energies provided by unfolding experiments showed that the coiled-coil
formed by the 1013-1056 peptide is more stable in GdnHCl than in
urea. The difference of free energy between the two denaturants could
be explained by intra- or interhelical destabilizing electrostatic
interactions arising in the peptide.
G
, were found by thermal and urea unfolding,
respectively, for the 1013-1056 peptide of human topoisomerase II
. Remarkably, the above values are similar to those recently
determined (-7.6 kcal/mol) for the yeast DNA topoisomerase II
entire molecule from sedimentation equilibrium in the analytical
centrifuge(32) . However, conditions adopted in experiments
with the peptide and the protein are different.
1013-1056 fragment associates into a
stable two-stranded
-helical coiled-coil structure. The dimeric
state of the fragment appears to be stabilized mostly through
hydrophobic interactions. The sensitivity reflected by the
-helix
against peptide concentration shows that coiled-coil formation plays a
non-negligible role toward the folding and stability of the fragment.
G
, which is found to be approximately the
same for the 1013-1056 fragment and the entire yeast
topoisomerase II (32) . Notwithstanding the particular
experimental conditions used for the fragment, we are tempting to
speculate on the involvement of 1013-1056 segment as a unique
region for the dissociation/reassociation process of topoisomerase II,
outward interactions with DNA. This proposal is consistent with the
results of Caron et al.(29) , which have pointed out
that topoisomerase II dimerization arises in the C-terminal half of
the protein and is further supported by the fact that this dimerization
would occur in a strongly conserved region of topoisomerase II.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.