Báo cáo khoa học: Inhibitor-mediated stabilization of the conformational structure of a histone deacetylase-like amidohydrolase pptx

Inhibitor-mediated stabilization of the conformational
structure of a histone deacetylase-like amidohydrolase
Stefanie Kern
1
, Daniel Riester
2
, Christian Hildmann
2
, Andreas Schwienhorst
2
and
Franz-Josef Meyer-Almes
1
1 Department of Chemical Engineering and Biotechnology, Darmstadt University of Applied Sciences, Germany
2 Institut fu
¨
r Molekulare Genetik und Praeparative Molekularbiologie, Institut fuer Mikrobiologie und Genetik, Goettingen, Germany
Nucleosomal histones are subject to a variety of
post-transcriptional covalent modifications, including
acetylation, methylation, phosphorylation and ubiquiti-
nation [1]. Reversible histone acetylation has been
shown to facilitate access of the transcriptional machin-
ery to DNA by disruption of nucleosome–nucleosome
and nucleosome–DNA interactions [2–4]. Acetylation of
histone proteins occurs at the e-amino group of lysine
residues near the N-termini of these proteins. The
steady-state histone acetylation level is the result of
opposing actions of histone acetyltransferases and his-
tone deacetylases (HDACs). In particular, HDACs are
promising therapeutic targets on account of their
involvement in regulating genes involved in cell cycle

Keywords
FB188; HDAH; histone deacetylase; protein
denaturation; protein stablization
Correspondence
F J. Meyer-Almes, Department of Chemical
Engineering and Biotechnology, University
of Applied Sciences Darmstadt,
Schnittspahnstr. 12, 64287 Darmstadt,
Germany
Fax: + 1649 6151168404
Tel: + 1649 6151168406
E-mail:
Website: />(Received 24 January 2007, revised 11 May
2007, accepted 15 May 2007)
doi:10.1111/j.1742-4658.2007.05887.x
Histone deacetylases are major regulators of eukaryotic gene expression.
Not unexpectedly, histone deacetylases are among the most promising tar-
gets in cancer therapy. However, despite huge efforts in histone deacetylase
inhibitor design, very little is known about the impact of histone deacety-
lase inhibitors on enzyme stability. In this study, the conformational stabil-
ity of a well-established histone deacetylase homolog with high structural
similarity (histone deacetylase-like amidohydrolase from Bordetella ⁄ Alcali-
genes species FB188) was investigated using denaturation titrations and
stopped-flow kinetics. Based on the results of these complementary approa-
ches, we conclude that the interconversion of native histone deacetylase-like
amidohydrolase into its denatured form involves several intermediates
possessing different enzyme activities and conformational structures. The
refolding kinetics has shown to be strongly dependent on Zn
2+
and to a

lesser extent on K
+
, which underlines their importance not only for cata-
lytic function but also for maintaining the correct conformational structure
of the enzyme. Two main unfolding processes of histone deacetylase-like
amidohydrolase were differentiated. The unfolding occurring at submolar
concentrations of the denaturant guanidine hydrochloride was not affected
by inhibitor binding, whereas the unfolding at higher concentrations of
guanidine hydrochloride was strongly affected. It was shown that the
known inhibitors suberoylanilide hydroxamic acid and cyclopentylpropio-
nyl hydroxamate are capable of stabilizing the conformational structure of
histone deacetylase-like amidrohydrolase. Judging from the free energies of
unfolding, the protein stability was increased by 9.4 and 5.4 kJÆmol
)1
upon
binding of suberoylanilide hydroxamic acid and cyclopentylpropionyl
hydroxamate, respectively.
Abbreviations
CypX, cyclopentylpropionyl hydroxamate; DG
u
, free energy of unfolding; Gdn-HCl, guanidine hydrochloride; HDAC, histone deacetylase;
HDAH, histone deacetylase-like amidohydrolase from Bordetella ⁄ Alcaligenes FB188; m
eq
, equilibrium parameter which reflects the
difference between the exposed surfaces and intermediate I and unfolded state D; SAHA, suberoylanilide hydroxamic acid.
3578 FEBS Journal 274 (2007) 3578–3588 ª 2007 The Authors Journal compilation ª 2007 FEBS
control [5,6]. To date, several HDAC inhibitors show
potency as antitumor agents, with several drug candi-
dates currently in phase I–III clinical trials [7]. Eukary-
otic histone deacetylases, as well as their bacterial

homologs, have been grouped into four classes, pri-
marily based on sequence similarity [8]. Whereas
class 3 enzymes (also termed ‘sirtuins’) are NAD-
dependent, class 1, 2 and 4 HDACs are zinc-dependent
hydrolases [9]. To date, crystal structures of two
class 1 enzymes and one class 2 enzyme, as well as of
enzyme–inhibitor complexes thereof, are known
[10–13]. However, no information is currently available
on the conformational stability of these enzymes. Fur-
thermore, despite the identification of a large number
of HDAC inhibitors, the effect of inhibitor binding on
enzyme structure and conformational stability of the
enzyme has not been analyzed in detail.
In general, the stability of proteins is an issue of
utmost interest in biochemistry and biophysics as well
as in industrial enzyme applications. The conforma-
tional stability of most proteins is surprisingly low,
generally between 20 and 60 kJÆmol
)1
[14,15]. This
small overall stability is the result of large contri-
butions from several important converse forces. The
major destabilizing force is conformational entropy.
The major stabilizing forces are hydrogen bonding and
the hydrophobic effect, which is also responsible for
the large change in heat capacity between the unfolded
and folded conformations [16,17].
For technical applications of enzymes, in most cases
maximal stability is desired without losses in activity.
There are mainly two approaches to stabilize proteins

(a) changes in amino acid sequence or (b) specific
binding of ions or compounds to the folded conforma-
tion. The largest increase in conformational stability
resulting from a single change in amino acid sequence
is the Asn57>Ile mutant of yeast iso-1-cytochrome c
by 17.64 kJÆmol
)1
[18]. Some studies report the stabil-
izing effects of inorganic ions that specifically bind to
the folded conformation of a protein [19–22]. For
example, Brandts et al. used differential scanning
calorimetry to measure protein stabilization by ferric
ions [23] and highly charged ligands. They showed that
binding of cytidine 2¢-monophosphate, other nucleo-
tide monophosphates, pyrophosphate and phosphate
shifted the transition temperature for ribonuclease
thermal unfolding [24]. In this study, Brandts et al.
suggested to use this approach for screening drug can-
didates for the estimation of binding constants or
screening solution conditions to optimize liquid protein
formulations with respect to stability. Recently, small
molecules were found to rescue mutant proteins from
degradation and to facilitate trafficking to their site of
action [25–27]. These compounds are called chemical
chaperones and those compounds which act selectively
on a certain pharmaceutical target protein are called
pharmacological chaperones. Although the precise
mechanism of action is not yet completely understood,
it is generally assumed that chemical chaperones stabil-
ize a protein conformation capable of escaping the

quality control system of the cell [25–27]. However, in
most of these studies the stabilization of the protein
conformation was not measured directly and quantified
in terms of free energy.
Here, we studied the conformational stability of the
HDAC class 2 homolog FB188 HDAH, a bacterial
HDAC-like amidohydrolase from Bordetella ⁄ Alcali-
genes species FB188 [28]. FB188 HDAH has been
shown to be an excellent model system for HDACs,
concerning both structure [13] and function [29,30].
The main focus of this report was to investigate the
impact of HDAC inhibitors as potential chemical
chaperones (i.e. stabilizers) as well as zinc and potas-
sium ions on the conformational stability of HDAH.
Two main denaturation phases of HDAH were differ-
entiated. The denaturation occurring at submolar con-
centrations of the denaturant guanidine hydrochloride
(Gdn-HCl) was not affected by inhibitor binding,
whereas the denaturation at higher concentrations of
Gdn-HCl was strongly affected. The existence of at
least one conformational intermediate was confirmed
by the fact that denaturation of HDAH occurs at a
slightly higher denaturant concentration than the loss
of enzyme activity. Moreover, the investigation of the
denaturation and refolding kinetics supports the view
that the interconversion between the native and the
completely denatured state of HDAH follows a
considerably complex mechanism. We have shown that
the overall conformational stability of HDAH is
significantly increased upon binding of the inhibitors

cyclopentylpropionyl hydroxamate (CypX) and sube-
roylanilide hydroxamic acid (SAHA). Data of refold-
ing kinetics demonstrate the strong stabilizing impact
of zinc ions, and, to a lesser extent of potassium ions,
on the conformational structure of HDAH.
Results and Discussion
Stabilization of conformational structure of
HDAH by inhibitors
Taking FB188 HDAH as a model of HDACs, we were
interested to see whether small-molecule inhibitors
would also act as molecular chaperones. To elucidate
the molecular mechanism of stabilization of protein
structure by inhibitor binding, we performed titrations
S. Kern et al. Inhibitor-mediated stabilization of HDAH
FEBS Journal 274 (2007) 3578–3588 ª 2007 The Authors Journal compilation ª 2007 FEBS 3579
using Gdn-HCl as the denaturant and analyzed
stopped-flow kinetics of the denaturation reaction as
well as refolding of HDAH in the absence and the
presence of small organic molecule inhibitors. Denatur-
ation experiments were performed in the presence of
0–4.5 m Gdn-HCl. HDAH showed a biphasic denatur-
ation curve upon increasing the concentration of the
denaturant (Fig. 1). The protein fluorescence excited
at 295 nm and measured at 350 nm originates from
five tryptophans of HDAH. One of the tryptophans
(Trp13) is solvent accessible. Two tryptophans
(Trp179, Trp191) are buried in the hydrophobic center
of the protein, and two further tryptophans (Trp7,
Trp192) have limited solvent accessibility. The trypto-
phans with no or limited solvent accessibility presuma-

bly possess relatively high quantum yields. Upon
unfolding, these tryptophans become more exposed to
water, which leads to a decrease of their quantum yield
and a shift of the maximum of the emission spectrum
from 353 nm in the native state to 360 nm in the dena-
tured state. The emission maximum at 353 nm did not
change at concentrations of Gdn-HCl < 1.5 m.In
contrast, an unusual decrease in HDAH protein fluo-
rescence intensity was observed at submolar concentra-
tions of Gdn-HCl. This decrease in fluorescence was
not caused by an artificial contribution of the Gdn-
HCl solution used in all denaturation experiments, as
confirmed in a control experiment where the dissolved
amino acid tryptophan was titrated with Gdn-HCl
(data not shown). Therefore, we conclude that partial
unfolding of HDAH takes place at submolar
concentrations of Gdn-HCl. This denaturation phase
at low denaturant concentration contributes about
40% to the overall process. This study concentrates
mainly on the denaturation effect at higher Gdn-HCl
concentrations, which, in contrast to the unfolding at
submolar concentrations of Gdn-HCl, is clearly affec-
ted by inhibitor binding. The complete denaturation
curve can be fitted using a model function consisting
of two addends. As the denaturation curve at submo-
lar denaturant concentration cannot be explained by a
simple two-state model where the native state denatur-
ates into an intermediate, the first addend just des-
cribes the shape of the denaturation phase at submolar
denaturant concentration and does not yield parame-

ters with thermodynamic meaning. However, the
second addend describing the major part of the
denaturation curve which is affected by binding of
inhibitors directly yields the free energy of unfolding,
DG
u
, of the intermediate in the absence of denaturant
and the equilibrium m
eq
value. m
eq
is a parameter
which reflects the change in compactness of HDAH
upon denaturation. The parameter is proportional to
the surface area buried in the intermediate state I.
Therefore, the DG
u
of 17.9 kJÆmol
)1
for the major
denaturation phase at higher Gdn-HCl concentrations
is a lower estimate of the overall conformational sta-
bility of free HDAH, being consistent with the con-
formational stability of most other proteins, which is
between 20 and 60 kJÆmol
)1
[14,15]. There are only
rare reports about conformational changes of protein
structures at submolar denaturation concentrations.
The structural changes of horseradish peroxidase and

spectrin at submolar concentrations of denaturant are
examples reported by Ray et al. [31] and Chakrabarti
et al. [32], although the observed changes in the bio-
physical parameters were much smaller as compared
with the denaturation of HDAH. Saturating concen-
trations of SAHA or CypX were used in all experi-
ments where inhibitors were present. The binding
constants of SAHA and CypX to HDAH were deter-
mined by Riester et al. [33], using a competitive bind-
ing assay based on fluorescence energy transfer, and
are summarized in Table 1. As only the denaturation
phase at higher denaturant concentration is affected by
the binding of inhibitors, the difference between the
free energy of protein unfolding of free and complexed
HDAH (DDG
u
) is identical to the increase of the con-
formational stability of HDAH by 9.4 and 5.4 kJÆ
mol
)1
upon binding of SAHA or CypX, respectively
(Table 1). This large contribution to the conformational
stability is at least one-third of the lower estimate of
the conformational stability of the whole protein. The
0
20
40
60
80
100

012345
c(Gdn-HCl) / M
Norm. Fluorescence Intensity
0
20
40
60
80
100
Enzyme Activity / %
- Inhibitor CypX
SAHA
Enzyme Activity
Fig. 1. Denaturation curves of 250 nM histone deacetylase-like
amidohydrolase from Bordetella ⁄ Alcaligenes FB188 (HDAH) in the
absence of inhibitor (squares) and in the presence of 100 l
M cyclo-
pentylpropionyl hydroxamate (CypX) (triangles) and suberoylanilide
hydroxamic acid (SAHA) (circles). The normalized fluorescence
intensity (excitation 295 nm, emission 350 nm) is plotted versus
the concentration of the denaturant guanidine hydrochloride (Gdn-
HCl). The unbroken lines (except that for enzymatic activity) are the
result of fitting the denaturation data to Eqn (1). The crosses
denote the corresponding relative enzyme activity of HDAH in the
absence of inhibitor.
Inhibitor-mediated stabilization of HDAH S. Kern et al.
3580 FEBS Journal 274 (2007) 3578–3588 ª 2007 The Authors Journal compilation ª 2007 FEBS
binding of the hydroxamic acid derivatives to the zinc
ion, His142, His143 and Tyr312 within the active site,
as well as hydrophobic interactions of the aliphatic

chains with Phe152 and Phe208, are believed to con-
tribute to the stabilization of the the major part of the
conformational structure of HDAH [13]. The m
eq
value of free and complexed HDAH is about )11 kJÆ
mol
)1
Æm
)1
. This is consistent with the assumption that
the intermediate, I, is more compact than the dena-
tured protein, D. The larger the m
eq
value, the greater
the difference between I and D in exposed surface
area. The magnitude of the m
eq
value is comparable to
the overall m
eq
values of other proteins, which range
between )2.5 and )18 kJÆmol
)1
Æm
)1
[34]. The part of
the protein that unfolds at submolar denaturant con-
centrations is quite labile and is not affected upon
inhibitor binding. To obtain more insight into the
mechanism of stabilization by HDAH inhibitors, the

refolding and denaturation kinetics in the presence and
absence of CypX were investigated. If not noted other-
wise, 10 mm K
+
was used in these experiments. The
refolding kinetics in the presence of 0.5 mm Zn
2+
and
100 lm CypX was slightly slower when compared with
the kinetics in the absence of CypX. At 50 lm Zn
2+
,
the overall refolding kinetics was markedly slower,
showing a sigmoidal increase. Upon the addition of
100 lm CypX, again the refolding kinetics was only
slightly slower than in the absence of CypX. This neg-
ative impact of CypX on refolding can be explained by
the Zn
2+
dependency of HDAH refolding. As pointed
out in the following section, the refolding kinetics is
strongly dependent on the concentration of zinc ions
(Fig. 2A,B). CypX is a hydroxamate derivative and hy-
droxamates are known to complex divalent cations,
such like Zn
2+
. Thus, free CypX is able to bind Zn
2+
ions, which otherwise would accelerate the refolding of
HDAH. Under these conditions of refolding, the com-

petition between HDAH and CypX for Zn
2+
binding
causes a slightly retarded refolding kinetics.
Table 1. Equilibrium parameters of histone deacetylase-like amido-
hydrolase from Bordetella ⁄ Alcaligenes FB188 (HDAH) in the
absence and the presence of inhibitors. K
2
denotes the binding
constant of the respective inhibitor to HDAH [33]. The free energy
of unfolding (DG
u
) from the intermediate to the denatured and the
parameter m
eq
were obtained from fitting the data of the equilib-
rium denaturation curve to Eqn (1). The increase in conformational
stability of HDAH, DDG
u
, upon inhibitor binding is the difference
between the free energy of unfolding of HDAH in the absence of
inhibitor and in the presence of 100 l
M of the noted inhibitor CypX
or SAHA.
Inhibitor
K
2
(10
6
ÆM

)1
)
m
eq
(kJÆmol
)1
ÆM
)1
)
DG
u
(kJÆmol
)1
)
DDG
u
(kJÆmol
)1
)
No inhibitor – ) 10.3 ± 1.6 17.9 ± 3.0 –
+ SAHA 1.0 ) 11.7 ± 1.5 27.3 ± 3.5 9.4
+ CypX 0.7 ) 10.4 ± 1.5 23.3 ± 3.6 5.4
0.8
1.0
A
B
C
0.6
0.4
0.2

0.0
0.8
1.0
0.6
0.4
0.2
0.0
0.8
1.0
0.6
0.4
0.2
0.0
0
10 20 30 40 50 60
Normalized FluorescenceNormalized Fluorescence Normalized Fluorescence
Time / s
Fig. 2. Refolding kinetics of histone deacetylase-like amidohydro-
lase from Bordetella ⁄ Alcaligenes FB188 (HDAH) (A) in the presence
of 10 m
M KCl and different concentrations of Zn
2+
(0 mM, blue;
0.05 m
M, red; 0.5 mM, dark green; 1.0 mM, black) and (B) in the
presence of different concentrations of Zn
2+
and K
+
ions (0 mM

Zn
2+
+0mM K
+
, brown; 0 mM Zn
2+
+10mM K
+
, blue; 1 mM
Zn
2+
+0mM K
+
, orange; 1 mM Zn
2+
+10mM K
+
, black) and (C) in
the presence of 10 m
M K
+
and in the presence of different concen-
trations of Zn
2+
ions in the absence or the presence of 100 lM
inhibitor cyclopentylpropionyl hydroxamate (CypX) (0.05 mM
Zn
2+
) CypX, red; 0.05 mM Zn
2+

+ Cyp X, magenta; 0.5 mM
Zn
2+
) CypX, dark green; 0.5 mM Zn
2+
+ CypX, light green). The
normalized fluorescence of stopped-flow experiments is plotted
versus time. First, the enzyme was denatured using 3
M guanidine
hydrochloride (Gdn-HCl). Then, refolding was initiated by diluting
the denatured enzyme in Tris buffer, pH 8.0, to a final Gdn-HCl con-
centration of 0.6
M.
S. Kern et al. Inhibitor-mediated stabilization of HDAH
FEBS Journal 274 (2007) 3578–3588 ª 2007 The Authors Journal compilation ª 2007 FEBS 3581
In contrast, CypX has a significant impact on the
denaturation kinetics of HDAH in 15 mm Tris ⁄ HCl
buffer, pH 8.0 (Fig. 3). At 2.8 m Gdn-HCl the ampli-
tudes of the denaturation curves were almost the same
in the absence and the presence of 100 lm CypX, but
the denaturation kinetics in the presence of CypX was
significantly slower (Fig. 3A). At 3.2 m, the denatura-
tion curves could not be distinguished (Fig. 3B). This
would be expected if the binding of CypX to HDAH
is inhibited in the presence of 3.2 m Gdn-HCl. This
would also explain why the effect of CypX on the
unfolding kinetics in the presence of 2.8 m Gnd-HCl is
weak compared with the strong effect of CypX on the
stability of the protein. Based on these results, we
conclude that the inhibitor CypX can stabilize the

conformational structure of HDAH by decelerating the
denaturation process. Hydroxamate-derived inhibitors
can even be contraproductive in refolding experiments
of Zn
2+
-dependent enzymes such as HDAH because
hydroxamate complexes Zn
2+
ions, which accelerate
refolding dramatically (see the next section).
Impact of Zn
2+
and K
+
ions on the refolding
of HDAH
The crystal structure of HDAH contains one Zn
2+
ion
at the bottom of the active site and two K
+
ions in
the neighbourhood of the active site [13]. The import-
ance of Zn
2+
and K
+
ions for the conformational
structure of HDAH was investigated by measuring the
refolding kinetics of HDAH (Fig. 2A,B) in the pres-

ence or absence of these cations.
The kinetics of HDAH refolding in the presence of
10 mm K
+
is strongly dependent on Zn
2+
, which
underlines the pivotal role of Zn
2+
within the active
site of HDAH for the conformational stability of
HDAH (Fig. 2A and 4A). If Zn
2+
is present at 0.5 or
1mm, the folding of the polypeptide chain into the
correct orientation of the enzyme is facilitated as a
result of the interactions between the zinc ion and the
adjacent amino acids Asp180, Asp268 and H182. The
kinetics in the presence of 0.5 mm Zn
2+
,10mm K
+
and a final concentration of 0.6 m Gdn-HCl at 21°C
behaves like a single exponential with a time constant
of 2.3 s. With decreasing concentrations of Zn
2+
, the
refolding kinetics becomes strongly retarded. At lower
Zn
2+

concentrations the kinetics changes into a sigmo-
idal behaviour, indicating a more complex mechanism
of the refolding process with at least one additional
intermediate in the absence or at lower concentrations
of Zn
2+
, which becomes rate limiting. Perhaps the
mechanism is followed also in the presence of
0.5 mm Zn
2+
, where the time course of the refolding
kinetics can be described by only one exponential. In
this case it could be assumed that the first process will
be accelerated by Zn
2+
, such that this step is no longer
rate limiting. Another explanation would be that the
refolding mechanism would change in the presence of
Zn
2+
. Refolding rates at 1 mm Zn
2+
, on the other
hand, gave rise to clear double-exponential decays with
two well-separated phases. Both effects – the slow
effect and the fast effect – strongly depend on the final
Gdn-HCl concentration (Fig. 4B). Such additional
effects might be ascribed to one of three phenomena:
(a) transient aggregation during folding [35,36] (b) cis-
trans isomerization (e.g. cis-trans isomerization about

prolyl-peptidyl bonds) [37–39] or (c) the formation or
decay of folding intermediates [40]. Aggregation can be
ruled out, as the refolding rate constant did not vary
significantly with protein concentration over a 10-fold
Normalized Fluorescence
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
1.2
B
A
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
0246810
0246810
Time / s
Time / s
Normalized Fluorescence
Fig. 3. Denaturation kinetics of 500 nM histone deacetylase-like
amidohydrolase from Bordetella ⁄ Alcaligenes FB188 (HDAH) in Tris-

phosphate buffer, pH 8.0, in the presence of 2.8
M (A) or 3.2 M (B)
guanidine hydrochloride (Gdn-HCl). The normalized fluorescence of
the intrinsic tryptophans of HDAH is plotted versus time. At the
lower concentration of Gdn-HCl (A) the difference between the
denaturation kinetics in the presence of 100 l
M cyclopentylpropio-
nyl hydroxamate (CypX) (red) and in the absence of inhibitor (blue)
becomes visible.
Inhibitor-mediated stabilization of HDAH S. Kern et al.
3582 FEBS Journal 274 (2007) 3578–3588 ª 2007 The Authors Journal compilation ª 2007 FEBS
range (data not shown). The fast phase, at
1mm Zn
2+
, is also much faster than a conventional
isomerization step, which usually has reaction rates in
the order of 10
)2
to 10
)4
Æs
)1
[41]. And, in contrast to
our observation, cis-trans isomerization processes do
not depend on the final denaturant concentration of
refolding. These arguments point again to at least one,
probably more, additional intermediates.
In the following, we will concentrate on the slow
phase and analyse the data according to the three-state
approach outlined in the experimental procedures.

Additional information about the accessible surface
of the conformational structures during the refolding
process can be obtained from the dependence of the
refolding rate constants on the final concentration of
Gdn-HCl [42,43].
Figure 4A shows plots of ln(k) of the slow refolding
process versus Gdn-HCl concentration at different
concentrations of Zn
2+
and 10 mm K
+
. In the
absence and the presence of 50 lm Zn
2+
at Gdn-HCl
concentrations below 0.6 m, the rate constants were
about one order of magnitude smaller compared with
the refolding kinetics in the presence of 0.5 and
1.0 mm Zn
2+
(Fig. 4A). At final Gdn-HCl concentra-
tions higher than 0.6 m, no temporal change in fluores-
cence intensity was observed in the presence of 0 or
50 lm Zn
2+
. This means that no measurable refold-
ing occurs at these concentrations of Zn
2+
and Gdn-
HCl. As seen inFig. 4A, the logarithmic refolding rate

constants in the presence of 0.5 and 1 mm Zn
2+
dis-
play a linear dependence at Gdn-HCl concentrations
higher than 1.0 m. This can be taken as a two-state
transition from the denatured to an intermediate state,
which dominates the folding reaction at concentrations
of > 1 m Gdn-HCl. The slope, which is proportional
to the difference between the accessible protein surface
after and before refolding, is negative, which is consis-
tent with the assumption that the intermediate is more
compact than the denatured protein. At concentrations
below 1 m Gdn-HCl, the plot of ln(k
obs
) versus denat-
urant concentration shows a clear rollover effect in
which the slope of the curve decreases significantly
(Fig. 4). This behaviour of the folding kinetics suggests
that an intermediate accumulates transiently during
refolding [44]. All curves of logarithmic rate constants
versus denaturant concentration could satisfactorily be
fitted to both the on-pathway or the off-pathway mod-
els (Eqns 7 and 9; Fig. 4). On the basis of the kinetic
data, it cannot be distinguished between productive
on-pathway intermediate I:
D ,
K
I
I ,
k

f
k
u
N;
and off-pathway intermediate C:
C ,
K
C
D ,
k
f
k
u
N:
The fitted parameters of both models are summarized
in Table 2. Further experiments are required to iden-
tify whether there is an on-pathway or an off-pathway
intermediate. Taking into account the sigmoidal shape
0.0
-4.0
-3.0
-2.0
-1.0
0.0
A
B
-4.0
-3.0
-2.0
-1.0

0.0
1.0
2.0
3.0
0.5 1.0 1.5 2.0 2.5
0.0 0.5 1.0 1.5 2.0 2.5
c(Gdn-HCl) / M
c(Gdn-HCl) / M
ln k
obs
ln k
obs
Fig. 4. Refolding rate constants of histone deacetylase-like amid-
ohydrolase from Bordetella ⁄ Alcaligenes FB188 (HDAH). The log-
arithm of the refolding rate constants is plotted versus the
concentration of guanidine hydrochloride (Gdn-HCl). The kinetics
were measured in the presence of 10 m
M KCl and the denoted
concentrations of ZnCl
2
. The data were fitted to a two-state model
(dashed line, Eqn 5) and to the on-pathway intermediate model
(solid line, Eqn 7). The kinetic parameters are summarized in
Table 2. (A) The concentrations of ZnCl
2
are 0 mM (open squares),
0.05 m
M (filled squares), 0.5 mM (circles) and 1.0 mM (diamonds).
Only the rate constant of the main slow increasing effect is shown
in panel A. (B) At 1 m

M Zn
2+
an additional fast effect with increas-
ing fluorescence intensity becomes visible. Both the rate constants
of the fast (triangles) and the slow (diamonds) processes are plot-
ted versus the concentration of Gdn-HCl.
S. Kern et al. Inhibitor-mediated stabilization of HDAH
FEBS Journal 274 (2007) 3578–3588 ª 2007 The Authors Journal compilation ª 2007 FEBS 3583
of refolding timecourses at very low Zn
2+
concentra-
tions (Fig. 2), the additional fast phase at 1 mm Zn
2+
(Fig. 4B) and the additional equilibrium denaturation
phase at low-denaturant concentration (Fig. 1), which
cannot be explained by a simple transition from the
native to one transition state, it is evident that folding
of HDAH is rather complex and must pass through
more than just one intermediate.
Without Zn
2+
and K
+
, no increase in fluorescence
can be detected within 60 s (Fig. 2B). In the presence
of 10 mm K
+
, but in the absence of Zn
2+
, a slow sig-

moidal increase in fluorescence intensity can be meas-
ured (Fig. 2B).
In summary, the refolding mechanism appears to be
strongly dependent on Zn
2+
and to a lesser extent on
K
+
, which underlines the importance of both cations,
not only for the function of the enzyme but also for the
correct conformational structure. There is strong
evidence that HDAH folding involves more than one
intermediate. It was shown that the known inhibitors
SAHA and CypX are capable of stabilizing the
conformational structure of HDAH. Judging from free
energies of unfolding, the conformational stability of a
complex between these inhibitors and HDAH is more
than 30% higher than the stability of unbound HDAH.
We suspect that hydroxamate-type HDAC-inibitors
such as CypX or SAHA not only hinder substrates to
obtain access to the active site, but rather may even
freeze HDACs in catalytically unproductive conforma-
tions. In this connection it is interesting to note that the
association kinetics of N-(2-furyl)acryloyl-hydroxamic
acid and HDAH can only be satisfactorily described by
a biphasic exponential model [31], suggesting a
multistep binding process, including conformational
changes of the enzyme. If we now assume a similar
behavior of eukaryotic HDACs upon inhibitor binding,
it is tempting to speculate that inhibitor-induced

conformational changes of HDACs are responsible
for breaking up corepressor complexes as, for example,
described in the case of acute myelocytic leucemia [45].
Furthermore, our results support the assumption that
specific ligands of proteins within cells may act as
molecular chaperones by stabilizing a protein confor-
mation capable of escaping the quality control system
of the cell. A better understanding of the impact of
inhibitor binding on the stability of target proteins
(e.g. HDAH) may result in new concepts for lead
structures.
Experimental procedures
Materials
His-tagged FB188 HDAH was prepared as described previ-
ously [28]. SAHA, CYPX and phenylpropionyl hydroxa-
mate were synthesized according to standard methods
[13,46,47]. If not stated otherwise, the denaturation experi-
ments were carried out in Tris-phosphate buffer consisting
of 250 mm sodium chloride, 250 lm EDTA, 15 mm Tris-
HCl and 50 m m potassium hydrogen phosphate, pH 8.0.
Enzyme activity assay
FB188 HDAH exhibits amidohydrolase [28] and esterase
activity [48]. Amidohydrolase activity was assayed in the
two-step assay [49,50]. As trypsin activity is required in this
type of assay, and trypsin rapidly denatures upon addition
of denaturant, the two-step assay was not suited for activity
measurements in samples containing Gdn-HCl. Esterase
activity was monitored using 4-methylcoumarin-7-acetate as
a substrate [48]. This type of assay was used for samples
containing Gdn-HCl.

Table 2. Kinetic parameters of histone deacetylase-like amidohydrolase from Bordetella ⁄ Alcaligenes FB188 (HDAH) refolding in the presence
of noted concentrations of Zn
2+
, c(Zn
2+
). Kinetic parameters of HDAH refolding are shown in the presence of 0.5 and 1.0 mM Zn
2+
. The
parameters were obtained by fitting the data of ln(k
obs
) versus the concentration of Gdn-HCl, c(Gdn-HCl), to different folding models (see
Eqns 5, 7 and 9). K
C
, ratio of off-pathway intermediate C and denatured state D; k
f
, the folding rate in the absence of denaturant; K
I
, ratio of
on-pathway intermediate I and denatured state D; m
f
reflects the change in solvent-accessible area in the process of refolding; m
i
, reflects
the change in solvent-accessible area in the transition from the denatured to the intermediate state.
c(Zn
2+
) Folding model k
f
(s
)1

) m
f
(kJÆmol
)1
ÆM
)1
) K
I
K
C
m
I
(kJÆmol
)1
ÆM
)1
)
0.5 m
M Two-state 9.3 ± 4.6 )8.6 ± 1.0 – – –
Three-state, on-path 0.38 ± 0.08 0.9 ± 1.3 152 ± 113 – )12.5 ± 0.7
Three-state, off-path 58 ± 51 11.7 ± 1.4 – 153 ± 112 )12.5 ± 0.7
1m
M Two-state 5.2 ± 1.6 ) 5.3 ± 0.5 – – –
Slow phase Three-state, on-path 0.39 ± 0,16 3.9 ± 2,6 26 ± 12 – )10.0 ± 2.1
Three-state, off-path 10.1 ± 6.0 )6.2 ± 0.8 26 ± 12 )10.1 ± 2.1
1m
M Two-state 34.5 ± 8.6 )5.9 ± 0.5 – – –
Fast phase Three-state, on-path 2.1 ± 4.9 13 ± 17 19 ± 37 – )20 ± 17
Three-state, off-path 41 ± 22 )6.3 ± 1.0 – 19 ± 37 )20 ± 17
Inhibitor-mediated stabilization of HDAH S. Kern et al.

3584 FEBS Journal 274 (2007) 3578–3588 ª 2007 The Authors Journal compilation ª 2007 FEBS
Denaturation experiments using guanidine
hydrochloride
In the denaturation experiments, 250 nm HDAH in phos-
phate buffer, pH 8.0, were titrated with increasing amounts
of a solution of 8 m Gdn-HCl in the same buffer. All
experiments were carried out at 21 ± 0.2 °C. The denatura-
tion of the protein was followed by measuring its trypto-
phan fluorescence. The tryptophans of HDAH were excited
at 295 nm and their fluorescence emission was measured at
350 nm in a Hitachi (Tokyo, Japan) F-4000 spectrofluo-
rometer using 5 and 10 nm slits, respectively. After each
addition of Gdn-HCl and thorough mixing, the fluores-
cence was measured until the signal was constant within ±
1% for at least 30 s. This value was considered to represent
the unfolding equilibrium and was plotted against the cor-
responding Gdn-HCl concentration. The resulting graph is
called the denaturation curve.
Data analysis of equilibrium protein denaturation
curves
The fitting of equilibrium unfolding curves is described in
detail by Santoro & Bolen [51] and directly gives thermo-
dynamic parameters of the corresponding denaturation
curves. The fitting function was slightly complemented to
account for the additional denaturation phase at submolar
denaturant concentrations. The fluorescence signal as a
function of denaturant concentration was fitted to the fol-
lowing expression:
F ¼
A

1 þ exphb Ãð½Gdn À HClÀIC
50
Þi
þ
F
I
þ F
D
exphÀðDG
u
þ m
eq
½Gdn À HClÞ=RTi
1 þ exphÀðDG
u
þ m
eq
½Gdn À HCl Þ=RTi
; ð1Þ
where DG
u
is the free energy of unfolding of an interme-
diate in the absence of denaturant; m
eq
is the equilibrium
m-value, which is proportional to the difference between
the exposed surfaces of intermediate I and unfolded state
D; F
I
and F

D
are the fluorescence signals of I and D;
A, b and IC
50
are the amplitude, the steepness and the
inflection point, respectively, and used as arbitrary
parameters to describe the contribution of the additional
denaturation process at submolar denaturant concentra-
tion; [Gdn-HCl] is the concentration of Gdn-HCl and
RT is the product of the gas constant and temperature.
All equilibrium and kinetic data were fitted using the
program scientist from micromath (St Louis, MO,
USA).
Stopped-flow kinetics
All measurements of displacement and renaturation kinetics
were carried out on a Bio-Logic (Claix, France) MOS-250
Stopped-Flow instrument equipped with a 150 W xenon
mercury light source attached to a manual monochromator
on an optical bench. The connection to the Bio-Logic
Stopped-Flow instrument was performed through a fiber
optic specially designed to match the stopped-flow cuvette
dimensions. The signal detection was performed by a pho-
tomultiplier directly mounted on the stopped-flow and con-
nected to its control unit. The photomultiplier was attached
at 90° of the light source allowing for fluorescence measure-
ments. The HDAH tryptophans were excited at 295 nm. A
polystyrene filter was installed in front of the photomulti-
plier tube to reject scattered light. The photomultiplier con-
trol unit was connected to a 16-bit A ⁄ D board installed in
a PC driven by the acquisition and analysis software

bio-kine32 (Claix, France).
The core unit of the instrument is a temperature-con-
trolled metal block containing three syringes and a mixing
chamber. The syringes are driven by precise and robust
high-speed stepping-motors. The dead time of the appar-
atus was calculated to be below 2 ms. The temperature was
controlled at 21 ± 0.2°C.
Stopped-flow data were fitted to either a monophasic
FðtÞ¼A
1
1 À exp À
t
s
1
 !
þ B ð2Þ
or a biphasic
FðtÞ¼A
1
1 À exp À
t
s
1
 !
þ A
2
1 À exp À
t
s
2

 !
þ B ð3Þ
exponential model by using a nonlinear least-square fit-
ting procedure integrated in the analysis software bio-
kine32. F(t) is the observed fluorescence of the protein at
time t after the start of the reaction and B is the back-
ground signal. A
1
and A
2
are the amplitudes of two
exponential changes, and s
1
and s
2
are their respective
kinetic time constants. The refolding kinetics were initi-
ated by mixing buffer consisting of 15 mm Tris ⁄ HCl,
pH 8.0, and denoted concentrations of Zn
2+
,K
+
ions or
CypX, and completely denatured HDAH dissolved in the
same buffer in the presence of 3 m Gdn-HCl. The dena-
turation kinetics were carried out by mixing 500 nm
HDAH (final concentration) in 15 mm Tris ⁄ HCl, pH 8.0,
in the absence or presence of 100 lm CypX and 15 mm
Tris ⁄ HCl buffer, pH 8.0, containing denoted concentra-
tions of Gdn-HCl.

Data analysis of refolding kinetics
The analysis of the kinetic data is based on a linear rela-
tionship between the log of microscopic rate constants and
the denaturant concentration. The following equations were
adapted from Mogensen et al. [52] and slightly modified to
fit rate constants determined from stopped-flow experi-
ments. Under the condition of the experiments the contri-
bution of unfolding could be disregarded.
S. Kern et al. Inhibitor-mediated stabilization of HDAH
FEBS Journal 274 (2007) 3578–3588 ª 2007 The Authors Journal compilation ª 2007 FEBS 3585
Two-state folding:
D ,
k
f
k
u
N ð4Þ
ln k
obs
¼ ln exp ln k
f
ÀÁ
þ m
f
Gdn À HCl½
ÀÁ
=RT
ÀÁ
ð5Þ
where in this simple two-state case D denotes the denatured

state and N denotes the native state of the protein; k
obs
is
the observed folding rate; k
f
is the folding rate in the
absence of denaturant and m
f
is the corresponding m-value
which reflects the change in solvent-accessible area in the
process of refolding. For a simple two-state folding mech-
anism a plot of ln k
obs
versus the concentration of Gdn-
HCl, c(Gdn-HCl), is expected to be linear over the whole
range of denaturant concentration. The accumulation of
on- or off-pathway intermediates during folding will give
rise to deviations from linearity, particularly at low denatu-
rant concentrations. The models for folding mechanisms
over in- or off-pathway intermediates follow.
(A) Folding over an on-pathway intermediate:
D ,
K
I
I ,
k
f
k
u
N ð6Þ

ln k
obs
¼ ln
exp ln k
f
ÀÁ
þ m
f
Gdn À HCl½=RT
ÀÁ
1 þ exp À ln K
I
ðÞþm
I
Gdn À HCl½=RTðÞðÞ
!
; ð7Þ
where I is an on-pathway intermediate between unfolded
and native protein and K
I
¼ [I] ⁄ [D] in the absence of denat-
urant.
(B) Folding with an off-pathway intermediate:
C ,
K
c
D ,
k
f
k

u
N ð8Þ
ln k
obs
¼ ln
exp ln k
f
ÀÁ
þ m
f
Gdn À HCl½=RT
ÀÁ
1 þ exp ln K
c
ðÞþm
I
Gdn À HCl½=RTðÞ
!
; ð9Þ
where C is an off-pathway folding intermediate and K
C
¼
[C] ⁄ [D] in the absence of denaturant.
Acknowledgements
This work was in part supported by grants to A.S.
(BioFuture 0311852 from the Bundesministerium fu
¨
r
Forschung und Technologie, Germany and Human
Frontier Science Program RGY0056 ⁄ 2004-C).

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Supplementary material

The following supplementary material is available
online:
Fig. S1. Fitting results of refolding kinetics at different
concentrations of zinc ions.
This material is available as part of the online article
from
Please note: Blackwell Publishing is not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corres-
ponding author for the article.
Inhibitor-mediated stabilization of HDAH S. Kern et al.
3588 FEBS Journal 274 (2007) 3578–3588 ª 2007 The Authors Journal compilation ª 2007 FEBS