Báo cáo khoa học: Structure–cytotoxicity relationships in bovine seminal ribonuclease: new insights from heat and chemical denaturation studies on variants ppt

Structure–cytotoxicity relationships in bovine seminal
ribonuclease: new insights from heat and chemical
denaturation studies on variants
Concetta Giancola
1
, Carmine Ercole
1
, Iolanda Fotticchia
1
, Roberta Spadaccini
2
, Elio Pizzo
3
,
Giuseppe D’Alessio
3
and Delia Picone
1
1 Department of Chemistry ‘Paolo Corradini’, University of Naples ‘Federico II’, Italy
2 Department of Biological and Environmental Sciences, Universita’ degli Studi del Sannio, Benevento, Italy
3 Department of Structural and Functional Biology, University of Naples ‘Federico II’, Italy
Keywords
calorimetric analysis; chemical denaturation;
cytotoxic ribonucleases; domain-swapping;
structure–activity relationships
Correspondence
D. Picone or C. Giancola, Dipartimento di
Chimica, Universita
`
di Napoli ‘Federico II’,
Complesso Universitario di Monte

Sant’Angelo, Via Cintia, 80126 Napoli, Italy
Fax: +39 081674409; +39 081674499
Tel: +39 081674406; +39 081674266
E-mail: ;

(Received 29 June 2010, revised 17
September 2010, accepted 25 October
2010)
doi:10.1111/j.1742-4658.2010.07937.x
Bovine seminal ribonuclease (BS-RNase), a homodimeric protein displaying
selective cytotoxicity towards tumor cells, is isolated as a mixture of two
isoforms, a dimeric form in which the chains swap their N-termini, and an
unswapped dimer. In the cytosolic reducing environment, the dimeric form
in which the chains swap their N-termini is converted into a noncovalent
dimer (termed NCD), in which the monomers remain intertwined through
their N-terminal ends. The quaternary structure renders the reduced pro-
tein resistant to the ribonuclease inhibitor, a protein that binds most ribo-
nucleases with very high affinity. On the other hand, upon selective
reduction, the unswapped dimer is converted in two monomers, which are
readily bound and inactivated by the ribonuclease inhibitor. On the basis
of these considerations, it has been proposed that the cytotoxic activity of
BS-RNase relies on the 3D structure and stability of its NCD derivative.
Here, we report a comparison of the thermodynamic and chemical stability
of the NCD form of BS-RNase with that of the monomeric derivative,
together with an investigation of the thermal dissociation mechanism
revealing the presence of a dimeric intermediate. In addition, we report that
the replacement of of Arg80 by Ser significantly decreases the cytotoxic
activity of BS-RNase and the stability of the NCD form with respect to
the parent protein, but does not affect the ribonucleolytic activity or the
dissociation mechanism. The data show the importance of Arg80 for the

cytotoxicity of BS-RNase, and also support the hypothesis that the reduced
derivative of BS-RNase is responsible for its cytotoxic activity.
Abbreviations
BS-RNase, bovine seminal ribonuclease; DSC, differential scanning calorimetry; GSH, glutathione; hA-BS-RNase, G16S ⁄ N17T ⁄ P19A ⁄ S20A
variant of bovine seminal ribonuclease; hA-mBS, G16S ⁄ N17T ⁄ P19A ⁄ S20A variant of the monomeric N67D variant of bovine seminal
ribonuclease with Cys31 and Cys32 linked to glutathione moieties; mBS, monomeric N67D variant of bovine seminal ribonuclease with
Cys31 and Cys32 linked to glutathione moieties; MxM, dimeric form of bovine seminal ribonuclease in which the chains swap their
N-termini; M=M, unswapped dimer of bovine seminal ribonuclease; NCD, noncovalent dimer; PDB, Protein Data Bank; RI, ribonuclease
inhibitor; RNase A, bovine pancreatic ribonuclease; S
80
-BS-RNase, R80S variant of bovine seminal ribonuclease; S
80
-hA-BS-RNase,
R80S ⁄ G16S ⁄ N17T ⁄ P19A ⁄ S20A variant of bovine seminal ribonuclease; S
80
-hA-mBS, R80S ⁄ G16S ⁄ N17T ⁄ P19A ⁄ S20A variant of the
monomeric N67D variant of bovine seminal ribonuclease with Cys31 and Cys32 linked to glutathione moieties; S
80
-mBS, R80S variant of
the monomeric N67D variant of bovine seminal ribonuclease with Cys31 and Cys32 linked to glutathione moieties.
FEBS Journal 278 (2011) 111–122 ª 2010 The Authors Journal compilation ª 2010 FEBS 111
Introduction
The outstanding feature of bovine seminal ribonucle-
ase (BS-RNase), proposed initially by Piccoli et al.,
(1992) [1] on the basis of biochemical data and struc-
turally proven a few years later [2,3], is the formation
of a dimeric form in which the chains swap their
N-termini (MxM). This phenomenon was later found
in many proteins, and is now well known under the
name of ‘3D domain swapping’. In most cases, the

swapping is associated with new biological functions,
and it has also been proposed as a possible mecha-
nism for protein aggregation in misfolding-associated
pathologies [4]. To date, more than 150 structures of
swapped proteins are present in the Protein Data
Bank (PDB). Among these, BS-RNase still represents
a special case, because the native protein is isolated as
a mixture of two dimeric isoforms, MxM and an
M=M [1], with or without the exchange of N-termini
respectively, in a molar ratio of about 2 : 1. Therefore,
only for this protein, the swapping is a physiological,
equilibrium process consisting of a dimer-to-dimer
interconversion; that is, it is not associated with varia-
tion in the quaternary structure. The swapping is
considered to be a prerequisite for most additional
biological properties accompanying the basal enzy-
matic activity, including a selective cytotoxic activity
towards malignant tumor cells [5]. However, the X-ray
structures of the two isoforms have revealed only
minor differences [2,3], located essentially at the loop
connecting the dislocated arms to the main body of
the protein. On the other hand, a so-called ‘buried
diversity’ [6] becomes evident when the protein is con-
sidered under different environments, such as cytosolic
reducing conditions.
In vitro, under mild reducing conditions, the two
interchain disulfides bridging the subunits of BS-RNase
undergo selective cleavage, so that M=M is converted
into two monomers, whereas MxM maintains a
dimeric structure, stabilized by noncovalent interac-

tions of the N-termini [1]. The monomeric form of
BS-RNase is readily neutralized by the ribonuclease
inhibitor (RI) [7], a protein that is abundant in mam-
malian cells, whereas the quaternary structure of the
reduced dimer, henceforth called the noncovalent
dimer (NCD), allows this protein to evade RI binding
[8]. It has been proposed that RI prevents endogenous
RNA degradation by binding monomeric ribonucleases
with very high affinity [9]. A schematic representation
of the different forms that BS-RNase can adopt in
different environments is given in Fig. 1, to emphasize
that, in the reducing conditions of the cytosol,
BS-RNase exists both as a monomer and as swapped
NCD stabilized by noncovalent interactions. NCD,
which is a transient species because, in solution, it dis-
sociates into two monomers, is considered to be
the form responsible for the cytotoxic activity of
the enzyme, given its resistance to RI. Furthermore, it
has been reported that the structural determinants
that favor the proper quaternary structure [6,10] and
the stability in solution of the swapped form play a
significant role in the additional biological properties
of the enzyme.
In this study, we have investigated the relationship
between the cytotoxic activity and the stability of the
NCD form of BS-RNase in comparison with variants
obtained by replacing the 16–20 hinge loop region
and ⁄ or Arg80 with the corresponding residues of
Structured digital abstract
l

MINT-8050499: BS-RNase (uniprotkb:P00669) and BS-RNase (uniprotkb:P00669) bind
(
MI:0407)bybiophysical (MI:0013)
l
MINT-8050482: BS-RNase (uniprotkb:P00669) and BS-RNase (uniprotkb:P00669) bind
(
MI:0407)byclassical fluorescence spectroscopy (MI:0017)
l
MINT-8050435: BS-RNase (uniprotkb:P00669) and BS-RNase (uniprotkb:P00669) bind
(
MI:0407)bycircular dichroism (MI:0016)
Fig. 1. Crystal structures of the multiple forms of BS-RNase: mBS,
PDB code 1N1X; M=M, PDB code 1R3M; MxM, PDB code 1BSR;
NCD, PDB code 1TQ9.
Stability–activity relationships in a cytotoxic dimeric ribonuclease C. Giancola et al.
112 FEBS Journal 278 (2011) 111–122 ª 2010 The Authors Journal compilation ª 2010 FEBS
bovine pancreatic ribonuclease (RNase A). It is
worth noting that none of these residues is actually
implicated in the catalytic activity. We have already
reported that neither substitution, i.e. the changes in
the 16–20 region or the change at Arg80, significantly
affects the swapping propensity of BS-RNase [11,12].
However, when the R80S mutation is inserted into the
construct containing the 16–20 hinge loop region of
RNase A [R80S⁄ G16S⁄ N17T⁄ P19A⁄ S20A variant of
BS-RNase (S
80
-hA-BS-RNase)], the MxM ⁄ M=M
molar ratio in the equilibrium mixture is changed from
2 : 1 to 1 : 2 [12]. On the basis of the hypothesis that

ascribes the special functions of BS-RNase to the
swapped form, in this study we investigated the biolog-
ical activity of this mutant, and found that it loses
almost all of the cytotoxic activity. Interestingly, the
single R80S substitution is sufficient to reduce the anti-
tumor activity almost to the same extent, leading us to
assign to this residue a pre-eminent role in the cyto-
toxic activity of BS-RNase, independently of the hinge
sequence. Furthermore, evaluation of the chemical and
thermal stability of the NCD forms of the variant pro-
teins in comparison with those of the parent one sup-
ports the hypothesis that the reduced swapped dimer
represents the bioactive form of BS-RNase.
Results
BS-RNase and its R80S variant (S
80
-BS-RNase), its
G16S ⁄ N17T ⁄ P19A ⁄ S20A variant (hA-BS-RNase) and
R80S/G16S/N17T/P19A/S20A variant (S
80
-hA-BS-RNase)
were expressed in monomeric form, with Cys31 and
Cys32 linked covalently and reversibly to two glutathi-
one (GSH) moieties, as already reported [12,13]. The
correctness of the fold of each monomeric protein was
confirmed by comparing their CD spectra and Kunitz
enzymatic activity on yeast RNA [14] with those of
parent monomeric BS-RNase (data not shown). Fur-
thermore, we compared the 2D NMR spectra of all
the monomeric variant proteins with that of the parent

monomeric N67D variant with Cys31 and Cys32
linked to GSH moieties (mBS), and found that the res-
onances of most amide signals are almost coincident,
the differences being essentially restricted to the back-
bone and side chain of the mutated residue(s) and of
those closest (in space). This is illustrated in detail for
the R80S variant of mBS (S
80
-mBS) in Fig. 2: panel
(A) shows the overlay of the
1
H-
15
N-HSQC spectrum
with that of mBS, and panel (B) gives a detail of the
3D structure of monomeric BS-RNase (PDB
code 1N1X), showing the local environment of Arg80.
It is very evident that the shifted residues belong to
the region encompassing Arg80 (78–84), to the hinge
(15 and 16) and to regions 45–49 and 101–103, which
are less than 4 A
˚
from the Arg80 side chain. The
overlay of the
1
H-
15
N-HSQC spectrum of mBS with
those of its G16S ⁄ N17T ⁄ P19A ⁄ S20A mBS and
R80S ⁄ G16S ⁄ N17T ⁄ P19A ⁄ S20A variants, indicated as

hA-mBS and S
80
-hA-mBS respectively, is shown
in Fig. S1.
Biological activity
The monomeric proteins were converted into dimers
by removal of the protecting GSH moieties and
15
N (p.p.m.)
1
H (p.p.m.)
A
B
Fig. 2. (A) Overlay of the
1
H–
15
N-HSQC spectra of the monomeric
derivatives of BS-RNase (black) and S
80
-BS-RNase (red) at 300 K.
Residues whose resonances are shifted are labeled. (B) Details of
the 3D structure of monomeric BS-RNase (PDB code 1N1X), show-
ing the local environment of Arg80. Residues that are less than 4 A
˚
from the Arg80 side chain are indicated.
C. Giancola et al. Stability–activity relationships in a cytotoxic dimeric ribonuclease
FEBS Journal 278 (2011) 111–122 ª 2010 The Authors Journal compilation ª 2010 FEBS 113
reoxidation of the intersubunit disulfides, followed by
incubation at 37 °C to allow the interconversion of

M=M and MxM to reach equilibrium. The cytotoxic
activity of the proteins towards tumor cells was mea-
sured by adding increasing concentrations (ranging
from 12.5 to 100 lgÆmL
)1
) of each variant to malig-
nant SVT2 cells, using BS-RNase as a positive control.
For a negative control, the proteins were also assayed
on nontumor 3T3 cell cultures at a final concentration
of 100 lgÆmL
)1
, and found to be nontoxic (Fig. S2).
The percentage of SVT2 cells surviving, illustrated in
Fig. 3, show that the replacement of Arg80 by Ser
induced a significant drop in cytotoxic activity, inde-
pendently of the hinge sequence. In contrast, we found
that changes in the 16–20 hinge region had only small
effects, as the cytotoxic activity of BS-RNase was only
slightly higher than that of hA-BS-RNase, and that of
S
80
-BS-RNase was very close to that of S
80
-hA-BS-
RNase.
Stability of NCD versus monomeric forms
In the search for the molecular basis for the induction
of the loss of cytotoxic activity of BS-RNase variants
reported in Fig. 3, we followed by CD the thermal
denaturation process of NCD derivatives, measuring

the molar ellipticity at 222 nm as a function of temper-
ature (Fig. 4A). As a comparison, the melting curves
of the corresponding monomers are reported in
Fig. 4B. The melting temperatures (T
m
values) of
NCD derivatives, which represent the midpoint of
the denaturation curve (Fig. 4A), were 59.4 °C for
BS-RNase and 59.0 °C for hA-BS-RNase, i.e. very
close to each other. In turn, significantly lower, and
comparable, T
m
values of 54.3 °C and 53.6 °C were
found for S
80
-hA-BS-RNase and S
80
-BS-RNase, respec-
tively. A similar trend was observed for the CD melting
temperatures of monomeric derivatives (Fig. 4B) and
[12], which can be separated into two groups, corre-
sponding to T
m
values around 58.0 °C and 54.0 °C for
the proteins with Arg80 or Ser80, respectively.
The CD melting curves of the monomers were used
to calculate the denaturation enthalpy changes by
using the van’t Hoff equation (Eqn 3 in Experimental
procedures), which describes two-state NMD transi-
tions. The data obtained, reported in Table 1, indicate

that DH
0
v:H:
values of the monomers follow the same
trend of T
m
values, with those of mBS and hA-mBS
close to each other and higher than the DH
0
v:H:
values
of S
80
-mBS and S
80
-hA-mBS, which, in turn, are close
to each other.
As a further step, we performed a calorimetric
analysis of all the proteins by standard differential
0
25
50
75
100
12.5 25 50 100
(µg·mL
–1
)
Cell survival (%)
Fig. 3. SVT2 cell survival after 48 h of incubation with different

amount of BS-RNase (
), S
80
-BS-RNase (h), hA-BS-RNase ( ) and
S
80
-hA-BS-RNase ( ).
10 20 30 40 50 60 70 80 90
0
1
A
B
Temperature (°C)
10 20 30 40 50 60 70 80 90
Temperature (°C)
Fraction unfolded
0
1
Fraction unfolded
Fig. 4. Thermal unfolding curves obtained following the change of
CD signal at 222 nm of NCDs (A) and monomeric derivatives (B) of
BS-RNase (
), hA-BS-RNase (x), S
80
-BS-RNase (•), and S
80
-hA-BS-
RNase (h). The unfolded fraction of protein was calculated as (Q –
Q
min

) ⁄ (Q
max
– Q
min
); Q is the ellipticity at 222 nm at a given temper-
ature, and Q
max
and Q
min
are the maximum and minimum values of
ellipticity corresponding to the denaturated state and native state of
proteins, respectively.
Stability–activity relationships in a cytotoxic dimeric ribonuclease C. Giancola et al.
114 FEBS Journal 278 (2011) 111–122 ª 2010 The Authors Journal compilation ª 2010 FEBS
scanning calorimetry (DSC) measurements. The calori-
metric profiles of all proteins are reported in Figs S3
and S4 for monomers and NCDs respectively. The
denaturation enthalpies of the monomeric derivatives
obtained from the DSC curves, reported as DH
0
cal
in
Table 1, were in good agreement with the van’t Hoff
enthalpies derived from DSC and CD curves, thus
confirming that the thermal denaturation process for
these proteins is a two-state transition process. An
inspection of the whole set of thermodynamic parame-
ters of monomeric forms, collected in Table 1, shows
that hA-mBS and mBS have comparable stabilities,
indicating that the substitution of four residues in the

hinge region does not significantly perturb the global
stability of the monomeric form of BS-RNase. On the
other hand, the single mutation R80S leads to
decreases of about 4 °C in the melting temperature
and of about 100 kJÆmol
)1
in the value of DH
0
cal
. This
destabilization is well reflected in DG
0
values, showing
that Arg80 is crucial for the stability of monomeric
form of BS-RNase.
For the NCD forms, thermal denaturation was
found to be an irreversible process, because there was
no refolding upon cooling of the protein solutions.
The irreversibility of the denaturation process does not
allow Gibbs energy calculations, but only a compari-
son of the melting temperatures and unfolding
enthalpy changes. DH
0
cal
and DH
0
v:H:
. values, both calcu-
lated from calorimetric profiles, are very similar and
the DH

0
cal
=DH
0
v:H:
ratio is in the range 0.98–1.07, sug-
gesting that the unfolding of the dimers is close to
being a one-step process (Table 1). The DH
0
v:H:
from
CD and DSC curves, relative to the unfolding of the
secondary and tertiary structure respectively, are for
each dimer very close, indicating simultaneous collapse
of both structures. As also indicated in Table 1, the
enthalpy changes for the NCD forms are very similar
to each other. For a comparison of the DH
0
cal
values of
the dimers with those of the corresponding monomers,
the enthalpy changes of NCD forms were calculated
at the melting temperatures of the corresponding
monomers, using the Kirchhoff equation. Values of
607 kJÆmol
)1
, 642 kJÆ mol
)1
, 581 kJÆ mol
)1

and 605
kJÆmol
)1
were obtained for NCD forms of BS-RNase,
hA-BS-RNase, S
80
-BS-RNase and S
80
-hA-BS-RNase,
respectively. All values are less than twice those of the
respective monomers. This indicates a loss of interac-
tions of the monomers in the dimeric structures. In
conclusion, all of the reported data indicate that the
R80S mutation is crucial for the loss in the enthalpic
content of NCDs of BS-RNase, engendering a lower
melting temperature of the R80S variants.
Urea denaturation of NCD forms
The conformational stability of the NCD forms
against the denaturing action of urea in comparison
with the corresponding monomers was investigated by
means of steady-state fluorescence and CD measure-
ments at pH 7.0.
Monomeric proteins showed sigmoidal transition
curves when the change in fluorescence intensity was
Table 1. Thermodynamic melting parameters of the unfolding process of monomers and NCDs of BS-RNase mutants. T
m
, denaturation
temperature; DH
0
(T

m
), calorimetric enthalpy change; DH
0
v:H:
, van’t Hoff enthalpy change; DC
0
p
ðT
m
Þ, excess heat capacity change; DS
0
(T
m
),
entropy change; DG
0
298
, denaturation Gibbs energy change at 298 K.
T
m
(°C)
DH
0
(T
m
)
(kJÆmol
)1
)
DH

0
v:H:
(kJÆmol
)1
)
DC
0
p
ðT
m
Þ
(kJÆmol
)1
ÆK
)1
)
DS
0
(T
m
)
(kJÆmol
)1
ÆK
)1
)
DG
0
298
(kJÆmol

)1
)
mBS 58.0 ± 0.5 428 ± 12 456 ± 20
408 ± 16
a
5.3 ± 0.5 0.72 ± 0.03 36.9 ± 5.5
hA-mBS 58.5 ± 0.5 405 ± 13 397 ± 21
420 ± 17
a
4.7 ± 0.6 0.69 ± 0.04 41.9 ± 6.3
S
80
-hA-mBS 53.9 ± 0.5 334 ± 10 330 ± 16
328 ± 13
a
4.8 ± 0.4 0.57 ± 0.03 24.9 ± 3.7
S
80
-mBS 54.5 ± 0.5 331 ± 9 325 ± 18
316 ± 13
a
5.4 ± 0.6 0.50 ± 0.03 25.4 ± 3.8
NCD BS-RNase 59.4 ± 0.5 619 ± 18 570 ± 23
528 ± 21
a
–––
NCD hA-BS-RNase 59.0 ± 0.5 646 ± 19 609 ± 20
588 ± 17
a
–––

NCD S
80
-hA-BS-RNase 54.3 ± 0.5 585 ± 17 597 ± 23
553 ± 22
a
–––
NCD S
80
-BS-RNase 53.6 ± 0.5 597 ± 18 590 ± 22
547 ± 21
a
–––
a
DH
0
v:H:
from CD measurements.
C. Giancola et al. Stability–activity relationships in a cytotoxic dimeric ribonuclease
FEBS Journal 278 (2011) 111–122 ª 2010 The Authors Journal compilation ª 2010 FEBS 115
recorded at the wavelength maximum, I
max
, as a func-
tion of urea concentration (insets in Fig. 5), whereas
the curves of NCDs displayed two transitions (Fig. 5).
The values of urea concentration at half-completion of
transition, C
½
, are shown in Table 2, which also
reports the values found by monitoring the molar ellip-
ticity at 222 nm with CD measurements; these reflect

conformational changes of the secondary structures for
monomers (insets in Fig. 6) and NCDs (Fig. 6). Also
in this case, two distinct C
½
values, the first value in
the 2–3 m range and the second close to the C
½
value
of the corresponding monomeric form (Table 2), were
observed for the dimers. To investigate in more detail
the mechanism of urea denaturation, we followed this
process at different protein concentrations, but focus-
ing on the parent BS-RNase and on the single-point
R80S variant. The results are reported in Fig. 7, where
the folded fraction is reported as a function of the urea
concentration at four different protein concentrations,
in the range 0.1–25 lm. According to Rumfeldt et al.
[15], the variation in the curve shape from sigmoidal to
biphasic observed when the protein concentration
increases confirms the presence of a dimeric intermedi-
ate in the dissociation process of both NCD variants.
The biphasic curves for the NCD forms of BS-RNase
and S
80
-BS-RNase at the highest concentration, where
the intermediate is present in significant amounts, were
analyzed according to the three-state equilibrium
model (N
2
MI

2
M2U) [16]. The following values for the
Gibbs energy changes and m-values were obtained:
DG
1
=14kJÆmol
)1
, m
1
=5kJÆmol
)1
Æm
)1
, DG
2
=80
kJÆmol
)1
, m
2
=19kJÆmol
)1
Æm
)1
for NCD BS-RNase;
and DG
1
=12kJÆmol
)1
, m

1
=6kJÆmol
)1
Æm
)1
, DG
2
=
43 kJÆmol
)1
, m
2
=15kJÆmol
)1
Æm
)1
for S
80
-BS-RNase.
The Gibbs energy values indicate that the perturbative
action of the urea is greater for the second transition,
I
2
M2U, than for the first transition, N
2
MI
2
, for both
dimers. The m-values also indicate that the surface
area exposed to solvent in the first transition is smaller

than that in the second transition. A comparison
between the two NCD forms shows that the R80S
mutation decreases the stability mainly in the step
I
2
M2U, and, if we assume that the final state is the
same for both NCD forms, this suggests that the R80S
mutation decreases the stability of the intermediate.
Structural models of the NCD forms
In the search for the possible origin of the reduced
activity of S
80
variants in both aggregation states, i.e.
in the monomeric and noncovalent swapped dimeric
forms, we examined the corresponding 3D structures.
All attempts to obtain crystals suitable for X-ray anal-
ysis of any form of the two S
80
-BS-RNase variants
had hitherto been unsuccessful. Supported by the
similarity of NMR spectra (Figs 2 and S1) of the
monomers, suggesting that the global architecture of
all the variant proteins is very similar to that of the
parent BS-RNase, and by the close similarity among
the X-ray structures of swapped isoforms of
hA-BS-RNase and BS-RNase [11], models of the 3D
structures of all proteins were obtained starting from
the X-ray structure of the corresponding form of the
parent BS-RNase, i.e. the monomeric derivative (PDB
02468

0.0
0.2
0.4
0.6
0.8
1.0
Fraction unfolded
[Urea] M
02468
0.0
0.2
0.4
0.6
0.8
1.0
Fraction unfolded
[Urea] M
02468
0.0
0.2
0.4
0.6
0.8
1.0
Fraction unfolded
[Urea]
M
02468
0.0
0.2

0.4
0.6
0.8
1.0
Fraction unfolded
[Urea]
M
AB
CD
Fig. 5. Urea-induced transition curves for
NCDs of BS-RNase variants and for the
corresponding monomers (inset), followed
by fluorescence spectroscopy.
(A) BS-RNase. (B) hA-BS-RNase.
(C) S
80
-BS-RNase. (D) S
80
-hA-BS-RNase.
The unfolded fraction represents the fraction
of denaturated protein, calculated as
(I ) I
min
) ⁄ (I
max
) I
min
); I is the fluorescence
intensity at a given temperature, and I
max

and I
min
are the maximum and minimum
values of fluorescence intensity
corresponding to the denaturated state and
native state of proteins, respectively.
Stability–activity relationships in a cytotoxic dimeric ribonuclease C. Giancola et al.
116 FEBS Journal 278 (2011) 111–122 ª 2010 The Authors Journal compilation ª 2010 FEBS
code 1N1X) and the noncovalent swapped dimer (PDB
code 1TQ9). A representation of the structural models
built for S
80
-hA-BS-RNase, which, among the variants
examined in this study, is the one hosting the highest
number of substitutions, is reported in Fig.8.
A careful inspection of these structures reveals that
all of the variant proteins examined, and notably both
of the S
80
variants, are characterized by the presence
of a decreased number of hydrogen bonds with respect
to the parent protein. This trend is even more evident
in the NCD derivatives: in these forms, all of the
mutants have fewer intersubunit hydrogen bonds
than the native protein. In particular, focusing on
residue 80, a contact between the side chains of Arg80
and Ser18 is detectable only in parent BS-RNase. Fur-
thermore, the same protein and hA-BS-RNase are sta-
bilized by a contact between a core residue of one
subunit (Gln101) and the hinge residues of the other

subunit (Ser20 in the case of BS-RNase and Ser18 in
the case of hA-BS-RNase).
Discussion
The antitumor activity of dimeric ribonucleases relies
on their quaternary structure, which enables the pro-
teins to avoid inhibition by RI and provides good sta-
bility in solution. We have already shown that Pro19,
Leu28 and, possibly, Gly16 play a relevant role in the
cytotoxicity, because they ensure the correct quater-
nary assembly of the NCD derivative of BS-RNase
[17]. However, the hinge residues and Leu28 have a
synergistic effect, because to observe a drastic reduc-
tion of the cytotoxic activity they have to be replaced
simultaneously [6,18].
In contrast, the data reported in this article show
that the substitution of the whole hinge region induces
only a small reduction in the basal cytotoxic activity
of BS-RNase, as in the case of the single mutants
P19A and L28Q [6]. Surprisingly, the replacement of
Arg80 by Ser significantly reduces the cytotoxic activ-
ity, as both S
80
variants are less active than the parent
BS-RNase. On the one hand, this shows the impor-
tance of Arg80, although it is irrelevant for the cata-
lytic activity or the swapping extent of BS-RNase [12];
on the other hand, it indirectly confirms that the
Table 2. Urea-induced denaturation parameters of monomers and
dimers of BS-RNase mutants, monitored by fluorescence and CD
spectroscopy. [Urea]

1 ⁄ 2
values were the denaturant concentrations
at half-completion of the transition.
[Urea]
1 ⁄ 2
(M)
CD
[Urea]
1 ⁄ 2
(M)
Fluorescence
mBS 5.70 ± 0.20 5.70 ± 0.08
NCD BS-RNase 3.00 ± 0.06
5.60 ± 0.04
3.00 ± 0.06
5.70 ± 0.03
S
80
-hA-mBS 4.10 ± 0.15 4.20 ± 0.20
NCD S
80
-hA-BS-RNase 1.60 ± 0.05
4.50 ± 0.15
1.60 ± 0.20
4.60 ± 0.20
hA-mBS 5.31 ± 0.03 5.36 ± 0.15
NCD hA-BS-RNase 2.20 ± 0.04
5.60 ± 0.01
2.62 ± 0.03
5.64 ± 0.01

S
80
-mBS 4.76 ± 0.01 5.04 ± 0.03
NCD S
80
-BS-RNase 2.07 ± 0.02
5.12 ± 0.02
2.17 ± 0.03
5.09 ± 0.02
02468
0.0
0.2
0.4
0.6
0.8
1.0
Fraction unfolded
[Urea] M
02468
0.0
0.2
0.4
0.6
0.8
1.0
Fraction unfolded
[Urea] M
02468
0.0
0.2

0.4
0.6
0.8
1.0
Fraction unfolded
[Urea] M
02468
0.0
0.2
0.4
0.6
0.8
1.0
Fraction unfolded
[Urea] M
AB
CD
Fig. 6. Urea-induced transition curves for
NCDs of BS-RNase variants and for the
corresponding monomers (inset), followed
by CD spectroscopy. (A) BS-RNase. (B)
hA-BS-RNase. (C) S
80
-BS-RNase. (D)
S
80
-hA-BS-RNase. The unfolded fraction of
protein was calculated as (Q ) Q
min
) ⁄

(Q
max
) Q
min
); Q is the ellipticity at 222 nm
at a given temperature, and Q
max
and Q
min
are the maximum and minimum values of
ellipticity corresponding to the denaturated
state and native state of proteins,
respectively.
C. Giancola et al. Stability–activity relationships in a cytotoxic dimeric ribonuclease
FEBS Journal 278 (2011) 111–122 ª 2010 The Authors Journal compilation ª 2010 FEBS 117
exchange of the N-terminal arms in BS-RNase-like
proteins is not sufficient to elicit the antitumor activity
[10]. We are aware that, in principle, the substitution
of a basic residue on the protein surface might reduce
the cytotoxic activity, by affecting the electrostatic
interaction with the cell membrane and perhaps the
internalization process [19], but, as shown by Notomi-
sta [20], the side of BS-RNase with the strongest posi-
tive potential is the one hosting the N-termini, which
is located opposite to Arg80 (Fig. 8).
The observation of models built with the X-ray
structure of the reduced dimer of BS-RNase (PDB
code 1TQ9) as template indicate that all of the variants
maintain a quaternary structure very close to that of
the parent protein, but are characterized by weaker

interactions between subunits. This result is also in
agreement with thermodynamic data and thermal and
chemical dissociation, which indicate a lower stability
of the variant proteins with respect to BS-RNase, prin-
cipally for both Ser80 variants.
We also investigated the dissociation mechanism,
and found that it is not affected by the mutations
investigated here. The experimental data related to
thermal denaturation processes of both Ser80 variant
proteins can be interpreted by surmising a simple
two-step process, from native dimer to denatured
monomers, whereas the presence of biphasic chemical
denaturation profiles, exhibited by both fluorescence
and CD curves, suggests the existence of a thermody-
namically stable intermediate induced by urea. This
discrepancy is not unexpected, because the two dena-
turation processes are induced by different perturbing
agents, proceed through different mechanisms (in the
case of the thermal denaturation process, the interme-
diate state is not present in significant amounts) and
end with completely distinct denatured states [21]. It is
possible that the chaotropic effect of urea initially
causes a small perturbation of the secondary and ter-
tiary structures of the proteins (DG
1
for the N
2
MI
2
transition is smaller than DG

2
of the I
2
M2U transi-
tion), and urea then stabilizes the intermediate through
its hydrogen-bonding ability. Furthermore, the com-
parison of the values obtained for the first step indi-
cates that, in all variants examined, the C
½
value of
the first step of the denaturation process is decreased
with respect to that of the parent protein, indicating
02468
0.0
0.2
0.4
0.6
0.8
1.0
Fraction unfolded
[Urea] M
A
0
24
68
0.0
0.2
0.4
0.6
0.8

1.0
Fraction unfolded
[Urea] M
B
Fig. 7. Urea-induced transition curves of NCDs of BS-RNase (A)
and S
80
-BS-RNase (B), followed by fluorescence spectroscopy at
different protein concentrations: (•), 0.1 l
M;( ), 1 lM;(h), 7 lM;
(
), 25 lM.
A
B
Fig. 8. (A) Crystal structure of the NCD form of BS-RNase (PDB
code 1TQ9). (B) Homology model of S
80
-hA-BS-RNase. The Arg80–
Ser18 and Ser20–Gln101 hydrogen bonds, which are detectable
only in BS-RNase, are indicated.
Stability–activity relationships in a cytotoxic dimeric ribonuclease C. Giancola et al.
118 FEBS Journal 278 (2011) 111–122 ª 2010 The Authors Journal compilation ª 2010 FEBS
that the interactions between the hinge region and resi-
due 80 are involved in the early stages of the chemical
denaturation process. As a consequence, S
80
-hA-BS-
RNase is the most prone to unfolding.
Our data suggest a correlation between the cytotoxic
activity of BS-RNase and its derivatives and the

stability of the corresponding reduced swapped forms.
This is also in agreement with the finding that cyto-
toxic RNases are, in general, very stable enzymes, and
with the relationship between resistance to unfolding
and cytotoxic activity observed for different variants
of RNase A [22,23]. In conclusion, the enhancement of
the conformational stability of the NCD derivative
represents a good approach to increase the toxicity of
BS-RNase towards cancer cells. In addition, the find-
ing that the structure and stability of the dimeric inter-
mediate, shown by urea denaturation studies, play a
key role in the dissociation process suggests further
investigations of the dissociation mechanism that may
help in the design of new cancer chemotherapeutic
agents based on BS-RNase.
Experimental procedures
Protein samples
All of the experimental procedures for obtaining significant
amounts of BS-RNase and its variants starting from the cor-
responding pET-22b(+) plasmid cDNA have already been
described in detail elsewhere [12,13]. As in the previous stud-
ies, all of the constructs were coding for an Asp residue at
position 67, instead of an Asn as in the wild-type protein, to
avoid side effects caused by the spontaneous deamidation of
Asn67 [24,25]. All of the proteins were expressed in Escheri-
chia coli cells and purified in monomeric form, with Cys31
and Cys32 linked to two GSH molecules. Monomers with
Cys31 and Cys32 in the reduced form, prepared as described
previously [12], were either carboxyamidomethylated with
iodoacetamide [26], to obtain the monomeric proteins used

directly for analysis, or dialyzed against 0.1 m Tris ⁄ acetate
(pH 8.4) for 20 h at 4 °C, to obtain dimers. In both cases,
the last step of the purification procedure was gel filtration
chromatography on a G-75 column (75 · 3 cm).
Freshly prepared dimeric proteins were essentially made
by M=M isomers: they were incubated at 37 °C for at
least 72 h, to allow the mixture to reach equilibrium.
The protein concentration was measured by UV spectro-
photometry, assuming e = 0.5 (0.1%, 278 nm, 1 cm) for
monomers and e = 0.465 for dimers.
NCDs
The NCDs were prepared according to the protocol previ-
ously described by Piccoli et al. [1], from the exchanged
form of the corresponding dimer. The reduction of the
interchain disulfide bridges was confirmed by SDS ⁄ PAGE,
under nonreducing conditions. The NCD forms were kept
at 4 °C until used for kinetic or thermodynamic analyses.
Cytotoxicity studies
Cytotoxicity was evaluated by performing the 3-(4,5-dim-
ethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduc-
tion inhibition assay as described previously [27]. Simian
virus-40-transformed mouse fibroblasts (SVT2 cells) and the
parental nontransformed Balb ⁄ C 3T3 line (3T3 cells) were
obtained from the ATCC (Manassas, VA, USA). The cells
were plated on 96-well plates at a density of 2.5 · 10
3
cells
per well in 100 lL of medium containing BS-RNase or one
of its variants (12.5, 25, 50 or 100 lgÆmL
)1

), and incubated
for 24 and 48 h at 37 °C. Cell survival is expressed as the
absorbance of blue formazan measured at 570 nm [27] with
an automatic plate reader (Victor3 Multilabel Counter; Per-
kin Elmer, Shelton, CT, USA). Each curve reports the aver-
age of three independent assays. Standard deviations in all
assays were in the range of 5–10%.
DSC
DSC measurements were performed on a third-generation
Setaram micro-DSC instrument at 1 °CÆmin
)1
. DSC data
were analyzed with a previously described program [28]. The
excess heat capacity of the protein in solution in the sample
cell was measured against a reference cell filled with the buf-
fer solution in the temperature range of 4–80 °C. The excess
heat function hDC
0
P
i. was obtained after baseline subtraction,
assuming that the baseline is given by the linear temperature
dependence of the native state heat capacity. The denatur-
ation enthalpies, DH
0
(T
m
), were obtained by integrating the
area under the heat capacity versus temperature curves. T
m
is the melting temperature, and corresponds to the maximum

of each DSC peak. The entropy changes corresponding to
the thermal denaturation of the monomers, DS
0
(T
m
), were
determined by integrating the curve obtained by dividing the
heat capacity curve by the absolute temperature.
Thermodynamic analysis
The van’t Hoff enthalpies were obtained by DSC profiles,
utilizing the equation [29]:
DH
0
v:H:
¼ aRT
2
m
DC
0
P
ðT
m
Þ=DH
0
ðT
m
Þð1Þ
where a originates from the stoichiometry of the reaction
(a =4 or a = 6 for monomer or dimer denaturation,
respectively), T

m
is the maximum of the DSC peak,
DC
0
P
ðT
m
Þ is the value of the excess heat capacity function at
T
m
, and DH
0
(T
m
) is the calorimetric enthalpy calculated by
direct integration of the area under the DSC peak. For the
C. Giancola et al. Stability–activity relationships in a cytotoxic dimeric ribonuclease
FEBS Journal 278 (2011) 111–122 ª 2010 The Authors Journal compilation ª 2010 FEBS 119
monomers, the entropy changes, DS
0
(T
m
), were determined
by integrating the curve obtained by dividing the heat
capacity curve by the absolute temperature, and the dena-
turation Gibbs energies at 298 K were calculated by com-
bining the classical Kirchoff equations:
DG
0
ð298Þ¼DH

0
ðT
m
Þ
T
m
À 298
T
m

À DC
0
p
ðT
m
Þ
ðT
m
À 298Þþ298 DC
0
p
ðT
m
Þ ln
T
m
298

ð2Þ
NMR

Two-dimensional NMR spectra were acquired at 300 K on
on Bruker DRX600 spectrometer by using standard pulse
sequence libraries. For the natural abundance
1
H–
15
N-
HSQC spectra, the protein concentration was 2.5 mm in
95% H
2
O ⁄ 5% D
2
O (pH 5.65).
1
H chemical shifts are rela-
tive to the water signal at 4.70 p.p.m. at 300 K, and
15
N
chemical shifts were indirectly referenced to the
1
H chemi-
cal shifts according to gyromagnetic ratios [30].
CD spectroscopy
CD measurements were performed on a JASCO 715 CD
spectrophotometer equipped with a thermoelectrically con-
trolled cell holder (JASCO PTC-348) that allows measure-
ments at a controlled temperature. Quartz cuvettes with
0.1 cm optical pathlength were used. Unless otherwise
reported, the protein concentration was 0.2 mgÆmL
)1

. The
CD spectra were recorded from 250 to 200 nm at 4 °C, and
normalized by subtraction of the buffer spectrum. Molar
ellipticity per mean residue [h] in deg cm
2
Ædmol
)1
was calcu-
lated from the equation [h] = 100[h]
obs
⁄ lC, where [h]
obs
is
the ellipticity measured in degrees, l is the pathlength of the
cell (cm) and C is the protein molar concentration. CD
spectra were recorded with a response of 4 s, a 1.0 nm
bandwidth and 20 nmÆmin
)1
scan rates. Thermal denatur-
ation curves were recorded in temperature mode at 222 nm,
with heating of the protein solution from 4 °Cto80°C
and a scan rate of 1.0 °CÆmin
)1
. The enthalpy changes were
calculated using origin 7.5 to fit CD melting curves by the
van’t Hoff equation:
@ ln K
@ 1
=
TðÞ


P
¼À
DH
0
R
ð3Þ
where K is the equilibrium constant and R is the gas con-
stant.
The urea-induced transition curves at 4 °C were obtained
by recording the CD signal at 222 nm for each independent
sample. All of the measurements were performed after over-
night incubation of the samples at 4 °C. The values of urea
concentration at half-completion of transition, [urea]
1 ⁄ 2
,
were calculated with the Boltzmann equation of origin 7.0.
Fluorescence analyses
Intrinsic protein fluorescence was recorded with a Perkin
Elmer LS50B spectrofluorimeter equipped with a circulating
water bath. Unless otherwise stated, the protein concentra-
tion was 0.2 mgÆmL
)1
, corresponding to 14 lm for the
monomers and 7 lm for the dimers. The excitation wave-
length was set at 274 nm, and the emission was measured
between 250 and 400 nm. The spectra were recorded at 4 °C
with a 1 cm cell and a 10 nm emission slit width, and cor-
rected for background signal. The urea-induced transition
curves were obtained by recording the change in fluorescence

intensity at maximum wavelength as a function of denatur-
ant concentration. The maximum emission wavelength of
proteins was recorded at 303 nm. Measurements were per-
formed after overnight incubation of samples at 4 °C. The
values of urea concentration at half-completion of transi-
tion, [urea]
1 ⁄ 2
, were calculated as for the CD measurements.
The biphasic curves at 25 lm were analyzed according to
the three-state equilibrium model with a dimeric intermedi-
ate, N2MI2M2U, where N2 is the native dimeric state, I2
the dimeric intermediate and U the unfolded monomer,
respectively [16]. Fitting of the data was performed with the
matlab 5.3 package. The model gives the Gibbs energy
changes, DG
1
and DG
2
, and the values of m
1
and m
2
for
the N2MI2 and I2M2U transitions, respectively. The
m-value is a measure of the dependence of DG on denatur-
ant concentration [31], and is proportional to the amount
of protein surface area exposed upon unfolding [32].
Molecular modeling
The structures of monomeric and dimeric variants of
BS-RNase were calculated from the NMR and X-ray struc-

tures deposited at the PDB. In particular, the NMR struc-
ture of mBS (PDB code 1QWQ) was used for S
80
-mBS,
whereas for the dimeric NCD form three crystallographic
structures were used, corresponding respectively to the
NCD BS-RNase derivative (PDB code 1TQ9), to the
N-dimer of RNase A (PDB code 1A2W) and to the PM8
human pancreatic RNase variant (PDB code 1H8X). The
atomic coordinates of the above-mentioned protein struc-
tures were used as a template to predict the 3D structure of
the variants, using modeller 8v5 [33]. The quality of the
structural models was evaluated with modeller, using the
score of variable target function method [34]. Model analy-
ses were performed with molmol [35] and pymol [36].
Acknowledgements
C. Ercole is a fellow of the Department of Chemistry
‘Paolo Corradini’ of University of Naples ‘Federico
II’, supported by a training grant from the ‘Compag-
nia di San Paolo di Torino’. We thank T. Tancredi for
help with NMR spectra acquisition, L. Petraccone for
Stability–activity relationships in a cytotoxic dimeric ribonuclease C. Giancola et al.
120 FEBS Journal 278 (2011) 111–122 ª 2010 The Authors Journal compilation ª 2010 FEBS
help with thermodynamic analysis, and P. A. Temussi
for critical reading and suggestions. Financial support
from MIUR (FIRB RBNE03B8KK) is also acknowl-
edged.
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Supporting information
The following supplementary material is available:
Fig. S1. Overlay of the
1
H–
15
N-HSQC of the mono-
meric derivatives of: mBs (black), hA-mBS (magenta)
and S
80

-hA-mBS (green).
Fig. S2. Cytotoxicity of BS-RNase and its variants, all
assayed at a final concentration of 100 lgÆmL
)1
,on
3T3 cells after 48 h of incubation.
Fig. S3. DSC profiles of monomers of BS-RNase
variants: (A) mBS; (B) hA-mBS; (C) S
80
-mBS; (D) S
80
-
hA-mBS.
Fig. S4. DSC profiles of NCDs of BS-RNase variants:
(A) BS-RNase; (B) hA-BS-RNase; (C) S
80
-BS-RNase;
(D) S
80
-hA-BS-RNase.
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Stability–activity relationships in a cytotoxic dimeric ribonuclease C. Giancola et al.

122 FEBS Journal 278 (2011) 111–122 ª 2010 The Authors Journal compilation ª 2010 FEBS