Báo cáo khoa học: Role of the hinge peptide and the intersubunit interface in the swapping of N-termini in dimeric bovine seminal RNase pptx

Role of the hinge peptide and the intersubunit interface
in the swapping of N-termini in dimeric bovine seminal RNase
Carmine Ercole
1
, Francesca Avitabile
1
, Pompea Del Vecchio
1
, Orlando Crescenzi
1
, Teodorico Tancredi
2
and Delia Picone
1
1
Dipartimento di Chimica, Universita
`
di Napoli Federico II, Italy;
2
Istituto Chimica Biomolecolare del CNR, Napoli, Italy
Bovine seminal ribonuclease (BS-RNase) is the only known
dimeric enzyme characterized by an equilibrium between
two different 3D structures: MxM, with exchange (or
swapping) of the N-terminal 1–20 residues, and M¼M,
without exchange. As a consequence, the hinge region 16–22
has a different tertiary structure in the two forms. In the
native protein, the equilibrium ratio between MxM and
M¼M is about 7 : 3. Kinetic analysis of the swapping pro-
cess for a recombinant sample shows that it folds mainly in
the M¼M form, then undergoes interconversion into the
MxM form, reaching the same 7 : 3 equilibrium ratio. To

investigate the role of the regions that are most affected
structurally by the swapping, we expressed variant proteins
by replacing two crucial residues with the corresponding
ones from RNase A: Pro19, within the hinge peptide, and
Leu28, located at the interface between subunits. We
compared the structural properties of the monomeric forms
of P19A-BS-RNase, L28Q-BS-RNase and P19A/L28Q-BS-
RNase variants with those of the parent protein, and
investigated the exchange kinetics of the corresponding
dimers. The P19A mutation slightly increases the thermal
stability of the monomer, but it does not alter the swapping
tendency of the dimer. In contrast, the L28Q mutation sig-
nificantly affects both the dimerization and swapping pro-
cesses but not the thermal stability of the monomer. Overall,
these results suggest that the structural determinants that
control the exchange of N-terminal arms in BS-RNase may
not be located within the hinge peptide, and point to a crucial
role of the interface residues.
Keywords: bovine seminal ribonuclease; domain swapping;
proline; ribonuclease A; site-directed mutagenesis.
Bovine seminal ribonuclease (BS-RNase), the only dimeric
protein in the pancreatic-type ribonuclease family, is
characterized in solution by an equilibrium between two
different structures [1]: in the form dubbed MxM, the
N-terminal arms are exchanged, or swapped, between the
two identical subunits, whereas in the form indicated as
M¼M no swapping occurs. In the native protein, the
equilibrium ratio between MxM and M¼M is about 7 : 3.
The two identical subunits are linked through two disulfide
bridges between Cys31 and 32 of one subunit with Cys32¢

and 31¢, respectively, of the partner subunit. Each subunit
has 83% of the amino-acid sequence identical with that of
bovine pancreatic RNase A. In particular, both enzymes
exhibit active sites constituted by identical amino-acid
residues in the same sequence position. Beside ribonuclease
activity, BS-RNase is endowed with several additional
biological activities, such as allostery [2], cytotoxicity toward
malignant cells [3], immunosuppression and antispermato-
genesis [4]. Domain swapping in BS-RNase was found to be
determinant for all of these activities, which may suggest a
physiological role for this structural peculiarity.
A folded and stable monomeric derivative of BS-RNase
can be obtained by selective reduction of the dimeric protein
with a moderate excess of dithiothreitol, and stabilized by
either alkylation of the exposed thiol groups [5] or reaction
with glutathione [6]. All monomeric derivatives of
BS-RNase are catalytically more active than the native
dimeric enzyme, but they do not exhibit any allosteric
property and have nodetectable ÔspecialÕ biological action [7].
In a recent paper, we reported an NMR characterization
of the N67D variant of monomeric BS-RNase [8], hence-
forth called mBS. The mutation avoids sample heterogen-
eity arising from the spontaneous deamidation of Asn67 [9],
but it does not affect enzymatic activity. Comparison of the
solution structures, as well as specific NMR relaxation
experiments, indicated that the hinge region 16–22 is much
more flexible in mBS than in RNase A. However, this
region shows the greatest sequence difference from RN-
ase A: GNSPSSS in BS-RNase vs. STSAASS in RNase A.
As a consequence of its flexibility, the structure of this

segment is not well defined in the solution structure of mBS
(Fig. 1A). Moreover, owing to extensive overlap of diag-
nostic signals, we could not unequivocally assign trans
isomerism to Pro19. Mutagenic studies have shown that
Pro19 and Leu28, which in BS-RNase makes a hydrophobic
contact at the interface between the two subunits (Fig. 1B),
are two crucial residues in inducing dimerization and
swapping N-terminal arms in RNase A variants [10,11].
As the first step of a study aimed to investigate, through a
Correspondence to D. Picone, Dipartimento di Chimica, Universita
`
di Napoli Federico II, Via Cintia, 80126, Napoli, Italy.
Fax: + 39 081 674409, Tel.: + 39 081 674406,
E-mail:
Abbreviations: BS-RNase, bovine seminal ribonuclease; mBS, mono-
meric N67D BS-RNase; RNase A, bovine pancreatic ribonuclease;
DVS, divinyl sulfone.
(Received 1 August 2003, revised 2 October 2003,
accepted 7 October 2003)
Eur. J. Biochem. 270, 4729–4735 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03872.x
systematic mutagenic approach, the role played by the hinge
and interface regions in the swapping process, we prepared
BS-RNase variants by replacing Pro19 and Leu28 with the
corresponding residues from RNase A. Here we report a
characterization of monomeric P19A, L28Q and P19A/
L28Q variants of mBS carried out by 2D NMR, CD and
differential scanning microcalorimetry, and an investigation
of the kinetics of swapping of all variant dimers in
comparison with that of the parent protein.
Materials and methods

Construction of mBS mutants
Site-directed mutagenesis was performed by a megaprimer
PCR method [12] to produce the mutants coding for P19A-
mBS, L28Q-mBS and P19A/L28Q-mBS, starting from the
pET-22b(+) plasmid cDNA coding for the wild-type
enzyme which already carries the N67D mutation, to avoid
sample heterogeneity by spontaneous deamidation at the
Asn67 site [8].
PCR amplification was performed with an Eppendorf
Mastercycler amplifier. The forward flanking primer
sequence used in these experiments, 5¢-GAGTGCGGCC
GCAAGCTTGGGCTG-3¢, had an estimated T
m
of 82 °C.
The reverse flanking primer sequence, 5¢-ATATACA
TATGAAAGAAAG-3¢, had a calculated T
m
of 42 °C.
The mutagenic primers for each variant are: P19A (5¢-AGA
GCTGCT
AGCAGAGTTG-3¢) and L28Q (5¢-CACAT
CATC
CTGGTTGCAA-3¢) (nucleotides that represent
mutations are underlined). For the mutant P19A/L28Q-
mBS the mutagenic primer L28Q was used starting from the
pET-22b(+) plasmid cDNA coding for the mutant P19A-
mBS. The amplified, mutated genes were separated, excised,
and purified from the agarose gel followed by cloning into
pET-22b(+) between the HindIII and NdeIsites.
Insertion of the correct mutations was confirmed by

DNA sequencing.
Recovery of proteins
All the proteins were expressed in Escherichia coli and
purified in monomeric form, with Cys31 and 32 linked to
two glutathione molecules, as described previously [13].
Monomers with Cys31 and 32 in the reduced form were
obtained by selective reduction of the mixed disulfide
bridges with a 5 : 1 molar excess of dithiothreitol for 20 min
at room temperature in 0.1
M
Tris/acetate buffer, pH 8.4.
The samples were either carboxyamidomethylated with
iodoacetamide [5], to obtain the monomeric proteins used
for CD and microcalorimetric analysis, or dialyzed against
0.1
M
Tris/acetate, pH 8.4, for 20 h at 4 °C, to obtain
dimers. The last step of the purification procedure was
always a gel filtration on Sephadex G-75 to separate
monomers from dimers. All dimerization steps were
performed at 4 °C.
Recombinant RNase A was obtained and purified as
described previously [8].
Protein homogeneity was verified by SDS/PAGE
and MALDI-TOF MS, registered at the Sezione di
Fig. 1. Ribbon representation of the solution
structure of mBS-RNase (A), as derived from
heteronuclear NMR data (pdb accession code
1WQW), and the X-ray structure of the MxM
form of BS-RNase (B; pdb accession code

1BSR). Pro19 and Leu28 are highlighted. The
figure was drawn with
MOLMOL
software [25].
4730 C. Ercole et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Spettrometria di Massa of the CIMCF, Universita
`
degli
Studi di Napoli Federico II. Protein concentration was
measured by UV spectrophotometry.
Kinetics of interconversion of dimeric forms
To follow the interconversion kinetics, dimer samples were
incubated at 37 °C. At given times, aliquots were with-
drawn, the interchain disulfide bridges were selectively
reduced as described above [1], and the mixture was
chromatographed on an analytical Superdex 75 HR 10/30
column. The amount of MxM and M¼M was evaluated
quantitatively by integrating the peaks of dimer and
monomer, respectively.
Assessing the extent of the N-terminal swap
at equilibrium
Cross-linking experiments were performed using divinyl
sulfone (DVS) as a 10% solution in ethanol. The protein
(20 lg) in sodium acetate buffer (100 m
M
,pH5,100lL)
and DVS (1 lL of the 10% solution) was incubated at
30 °C [11]; this is  1000-fold excess of sulfone over each
subunit of the protein. Aliquots were withdrawn over a
period of 96 h, quenched with 2-mercaptoethanol (final

concentration 200 m
M
), incubated for 15–30 min at room
temperature, and loaded on gels for reducing SDS/PAGE.
The ratio of monomer to cross-linked dimer was estimated
qualitatively by Coomassie blue staining.
NMR
NMR measurements were performed on Bruker DRX400
and DRX500 spectrometers. All spectra were collected
using the standard Bruker pulse sequence library. Protein
concentration was 2 m
M
in 95% H
2
O/5% D
2
O, pH 5.65.
CD
The CD spectra were recorded with a Jasco J-715 spectro-
polarimeter equipped with a Peltier-type temperature con-
trol system (model PTC-348WI). The instrument was
calibrated with an aqueous solution of
D
-10-camphorsulf-
onic acid at 290 nm [14]. Molar ellipticity per mean residue,
[h] in degreesÆcm
2
Ædmol
)1
, was calculated from the equation

[h] ¼ h
obs
mrw/10lC,whereh
obs
is the ellipticity measured in
degrees, mrw is the mean residue molecular mass (117 Da
[5]), C is the protein concentration in gÆL
)1
,andl is the
optical path length of the cell in cm. A 0.1-cm path length
cell and a protein concentration of  0.3 mgÆmL
)1
in 10 m
M
sodium acetate buffer, pH 5.0, were used. CD spectra were
recorded at 25 °C with a time constant of 16 s, a 2-nm band
width, and a scan rate of 5 nmÆmin
)1
; they were signal-
averaged over at least five scans, and baseline-corrected by
subtracting the buffer spectrum. Thermal unfolding curves
were recorded in the temperature scan mode at 222 nm
from 25 °Cupto85°C with a scan rate of 0.5 KÆmin
)1
.
Scanning calorimetry
Calorimetric measurements were performed on a second-
generation Setaram Micro-DSC. A scanning rate of
0.5 °CÆmin
)1

was chosen for all experiments. The raw data
were converted into an apparent molar heat capacity taking
into account the instrument calibration curve and the
buffer–buffer scanning curve, and by dividing each data
point by the scan rate and the protein molar concentration
in the sample cell. Finally, the excess molar heat capacity
function, <DCp>, was obtained after baseline subtraction,
assuming as reference the heat capacity of the native state
[15].
Results
Recombinant mBS and its P19A, L28Q and P19A/L28Q
variants (P19A-mBS, L28Q-mBS and P19A/L28Q-mBS,
respectively), all with Cys31 and 32 linked to two glutathi-
one molecules, were obtained in pure form with a yield of
about 15 mgÆper L culture. Each of these variants retains a
catalytic activity against yeast RNA comparable with that
of parent mBS, indicating that a native conformation is
present. A further indication of the similarity of their global
fold to that of the parent protein is provided by the 1D
1
H-NMR spectra (data not shown), which display all the
characteristic signals in almost identical positions. The
similarity was confirmed by CD measurements (Fig. 2).
The estimation of secondary-structure content, performed
by the neural network-based procedure implemented in the
program
K
2
D
[16,17], yielded very similar values for all the

protein samples (28% a-helix, 36% b-sheet and 40%
random coil); these values are also in good agreement with
the secondary structure derived from the NMR structure of
mBS [8].
To allow a more accurate evaluation of the effect of single
point mutations on the solution structure of monomeric
derivatives, we analysed the 2D NMR spectra of the
different variants of mBS. Figure 3 shows the expanded
regions of TOCSY spectra of L28Q-mBS (panel L28Q),
P19A/L28Q-mBS (panel PALQ) and P19A-mBS (panel
P19A), in comparison with the same region of the parent
mBS (panel mBS). The new signal at 8.40–1.40 p.p.m.,
which appears in the spectra of P19A-mBS and
Fig. 2. Far-UV spectra of mBS-RNase (solid curve), P19A-mBS-RNase
(dashed curve), L28Q-mBS-RNase (dotted/dashed curve) and P19A/
L28Q-mBS-RNase (dotted curve) in 10 m
M
sodium acetate buffer,
pH 5.0, 25 °C. The horizontal dotted line indicates the zero value of the
ellipticity.
Ó FEBS 2003 Structural properties of BS-RNase variants (Eur. J. Biochem. 270) 4731
P19A/L28Q-mBS, has been attributed to Ala19 in spite of
the difference with the assignment made for RNase A in
very similar experimental conditions [18]. Moreover, in the
spectra of L28Q-mBS and P19A/L28Q-mBS, the correla-
tions that belong to Leu28 are missing, and are replaced by
a set of new signals tentatively attributed to Gln28 (data not
shown). Apart from these expected differences, the high
similarity among the spectra of the variants and the parent
protein provides additional, strong evidence of an essentially

identical fold of all variant proteins. However, the
NH-C
b
H
3
cross-peak of Ala19 is significantly broader than
other cross-peaks; this may reflect an equilibrium between
different conformations of the 16–22 hinge region, occurring
at a rate comparable to the NMR chemical shift time scale.
A thermodynamic characterization of all monomeric
proteins was performed by differential scanning calori-
metry. The results, shown in Table 1, agreed with the
temperature-induced unfolding curves obtained by CD
measurements (data not shown). No sizeable differences
were found among the monomeric variants tested, and only
for P19A-mBS a significant thermal stabilization occurs.
The introduction of Ala for Pro19 makes mBS more similar
to RNase A, which has a thermal stability higher than that
of mBS. However, P19A/L28Q-mBS has the same thermal
stability of the parent protein, in spite of greater similarity to
RNase A.
To directly characterize the process of exchange of the
N-termini, we prepared dimers of all variants. The mono-
meric proteins were submitted to mild reduction, which
selectively removes the glutathione molecules linked to
Cys31 and 32, followed by air oxidation and gel filtration on
Sephadex G-75 to obtain the corresponding dimers. We
obtained 80% of dimer for both BS-RNase and P19A-BS-
RNase, whereas in the case of L28Q and P19A/L28Q
variants the yield of dimer was significantly lower,  50%.

The recombinant dimers were obtained predominantly in
their nonswapped forms; the extent of exchange of the
N-termini, as evaluated by selective reduction of the
interchain disulfide bridges followed by gel filtration (vide
supra), was initially  15%. The dimers were then incubated
at 37 °C for a week, to allow equilibration between the
interconverting forms. A qualitative evaluation of the extent
of swapping at equilibrium was performed by cross-linking
experiments with DVS, followed by SDS/PAGE analysis
under reducing conditions. DVS covalently joins the two
His residues of the active site (His12 and His119) [11], which
belong to the same subunit in M¼ M and to different
subunits in MxM. Thus, upon reduction and denaturation,
either a product of molecular mass 27 000 Da (from MxM)
or a product of molecular mass 13 500 Da (from M¼M) is
obtained. Figure 4 depicts the time-dependent course of this
reaction in the equilibrium mixtures of dimers. After 24 h of
reaction, all proteins show almost the same relative
proportion of exchanging and nonexchanging forms.
However, after 48 h, a slight prevalence of the MxM form
can be seen in the BS-RNase and P19A variant, whereas the
L28Q and P19A/L28Q variants still contain comparable
amounts of MxM and M¼M. This becomes even more
Fig. 3. Regions of 500 MHz 2D TOCSY
spectra of L28Q-mBS-RNase (panel L28Q),
P19A/L28Q-mBS-RNase (panel PALQ),
P19A-mBS-RNase (panel P19A), and
mBS-RNase (panel mBS). The N
1
H/b-methyl

cross-peak of Ala19 is boxed in the P19A and
PALQ panels.
Table 1. Thermodynamic parameters of thermal denaturation of mBS,
P19A-mBS, L28Q-mBS, P19A/L28Q-mBS and RNase A. Estimated
error on Td, DdH and DdCp was 0.2 °C, 5% and 10%, respectively.
Td(°C)
DdH(Td)
(kJÆmol
)1
)
DdCp
(kJÆmol
)1
ÆK
)1
)
mBS 53.4 370 4.8
P19A-mBS 55.0 383 4.6
L28Q-mBS 53.0 360 7.5
P19A/L28Q-mBS 53.0 387 5.0
RNase A 58.8 423 4.1
4732 C. Ercole et al.(Eur. J. Biochem. 270) Ó FEBS 2003
evident when the reaction time is extended to 96 h: the
approximate 1 : 1 ratio for L28Q and P19A/L28Q variants
did not change, whereas a clear prevalence of the exchanged
form is present in native BS-RNase and P19A variant.
A more detailed comparison of the swapping process
among the different BS-RNase variants has been performed
by a different method, based on the observation that, after
selective reduction of the interchain disulfide bridges, the

MxM form retains a dimeric structure, whereas the M¼M
form dissociates into two monomers upon gel filtration [1].
We observed (Fig. 5A) that, at 37 °C, the dislocation of the
N-terminal arms for the recombinant BS-RNase is very
similar to that reported for the native enzyme [1], reaching
the same 7 : 3 equilibrium in  5 days. This result also
provides indirect evidence of correct pairing of the inter-
chain disulfide bridges in the recombinant sample [11]. The
kinetics of the N-terminal arms swapping in the P19A-BS-
RNase is comparable to that of BS-RNase (Fig. 5B).
Interestingly, the amount of the MxM form at the plateau
as evaluated by integrating the peaks obtained on gel
filtration is 70%, i.e. in complete agreement with that
obtained for the native enzyme and also in agreement with
the qualitative estimation based on the DVS reaction. In
contrast, Fig. 5C,D clearly shows that replacement of
Leu28 with Gln affects in a significant way the kinetics
and equilibrium of swapping in both the L28Q-BS-RNase
and P19A/L28Q-BS-RNase variants. Furthermore, the
plateau corresponds to 50% of MxM for both proteins, in
agreement with the DVS cross-linking results.
As reported previously [1], at 4 °C the interconversion of
the native protein was obviously slowed down. For instance,
after 500 h only 25% of MxM is present in BS-RNase, and
20% in P19A-BS-RNase, i.e. the process is still far from
equilibrium. To assess this hypothesis, we monitored the
reverse process, namely the interconversion of MxM into
M¼M, for P19A-BS-RNase. To isolate the MxM dimer,
the equilibrium mixture, containing both forms in the usual
7 : 3 ratio, was reduced selectively with dithiothreitol and

gel filtered. The fractions containing the reduced dimer were
pooled and air oxidized. The amount of the MxM form in
the isolated sample at this stage was 85%. Aliquots were
then separately incubated at 4 °Cand37°C, and the extent
of the swapping was assessed as previously described.
Figure 6 shows the amount of the MxM form present as a
function of incubation time. P19A-BS-RNase behaves like
the native sample [1], reaching the typical equilibrium
mixture in  180 h at 37 °C, whereas at 4 °C the process
seems kinetically frozen, and the content of MxM is
constant over several weeks.
Discussion
In this work, we have investigated the effects of mutation
of two paradigmatic residues involved in the dislocation
of BS-RNase N-terminal arms, i.e. Pro19 and Leu28,
located in the hinge peptide and in the intersubunit
interface, respectively. CD and NMR spectra suggest a
close similarity in the global fold and in the structural
properties of all monomeric variants to those of the
parent protein. These results are in agreement with the
solution structure of mBS [8], which indicates that both
residues are solvent exposed and make only a limited
number of van der Waals interactions with the rest of the
protein. The only difference related to the substitution of
Pro with Ala is a slight increase in the thermal stability,
which becomes more similar to that of RNase A. This
result is in agreement with theoretical calculations [19],
and CD and calorimetric studies on the A19P-RNase A
variant [20], which both support a destabilizing effect
resulting from the introduction of a Pro at position 19 in

RNase A.
Surprisingly, the substitution of Pro19 by Ala does not
affect the dislocation of N-termini in BS-RNase. A Pro
residue is often found in crucial regions of other domain
swapped proteins [21], and cis–trans Pro isomerization has
been regarded as a key event in protein folding (for a recent
overview, see Wedemeyer et al. [22]). This observation led to
the definition of hinge Pro residues as Ôquaternary structure
helpersÕ. The similarity of kinetic behaviour between
BS-RNase and P19A-BS-RNase seems to rule out the
involvement of a cis–trans Pro19 isomerization as a crucial
step in the swapping mechanism of BS-RNase. In the
crystallographic structure of BS-RNase MxM form [23], the
peptide bond between residues 18 and 19 has a trans
conformation, and it seems reasonable to assume that the
same holds for the P19A-BS-RNase MxM form. If Pro19
were cis in the M¼M form, the P19A variant would hardly
be expected to display such similar thermodynamic and
kinetic behaviour in the interconversion process, as a cis
peptide bond is significantly less stable for Ala than for Pro.
In other words, in the P19A M¼M form, Ala19 would
either be forced into an unfavourable cis conformation or
would have already assumed the intrinsically preferred trans
conformation; in both cases, an effect on the swapping
process would be expected.
Even from a structural point of view, the role of Pro19 is
still ambiguous. All structural studies on BS-RNase report
poor definition of the hinge region around Pro19, and this
abnormality has often been taken as an indication of high
Fig. 4. SDS/PAGE analysis of the DVS cross-

linking reaction of BS-RNase (lane 1), P19A-
BS-RNase (lane 2), L28Q-BS-RNase (lane 3)
and P19A/L28Q-BS-RNase (lane 4).
Ó FEBS 2003 Structural properties of BS-RNase variants (Eur. J. Biochem. 270) 4733
intrinsic flexibility of this tract of the protein. Yet, in the
crystallographic structure of a dimeric variant of human
pancreatic ribonuclease, called PM8 [24], in which the
N-terminal region and the whole hinge loop are identical
with those of BS-RNase, the hinge assumes a 3
10
helix
structure. This result suggests that the flexibility of the
corresponding region in BS-RNase cannot be ascribed to
the hinge loop itself, but probably arises from interactions
with other parts of the molecule.
Overall, our data indicate that Pro19 is not crucial in the
swapping process, and suggest that the structural determi-
nants for this process are located in regions different from
the hinge loop 16–22.
In the search for the region primarily involved in the
swapping, we focused our attention on the interface between
the subunits. In a previous study [8] we found that, in mBS,
helix 2 (residues 24–32) is more disordered than in
RNase A. This disorder may be attributed to the proximity
of the hinge peptide (residues 16–22), or possibly to an
unfavourable solvent exposure of the Leu28 side chain
(residue 28 is Gln in RNase A). As far as the monomer is
concerned, introduction of Gln for Leu at position 28 does
not increase the stability or significantly affect the structure,
in spite of the greater hydrophilicity and helical propensity

of Gln. However, the presence of Leu28 in BS-RNase seems
to favour the dimerization process: for both L28Q-BS-
RNase and P19A/L28Q-BS-RNase, the yield of dimer
obtained on oxidation of the monomeric, reduced protein is
only 50%, which is significantly lower than for BS-RNase
and P19A-BS-RNase (at least 80%). Interestingly, literature
data for some variants of RNase A also report a higher
yield of dimer when Gln28 is substituted with Leu11. The
influence of the Leu side chain is also evident during the
swapping process, and again the two mutants in which
Leu28 had been substituted display closely matching kinetic
behaviours and at equilibrium contain a smaller percentage
of MxM (50%). Thus, several data indicate that Leu28 is
crucial not only for the interaction between the subunits, but
even for the swapping process. Finally, the close similarity
between the swapping behaviour of the N-terminal arms in
L28Q-BS-RNase and P19A/L28Q-BS-RNase confirms that
Pro19 does not represent a key residue in this process.
From a different perspective, our results provide further
evidence of the evolutionary significance of the A19P and
Q28L substitutions in the path leading from RNase A to
Fig. 6. Kinetic analysis of the MxM to M=M conversion for P19A-BS-
RNase. The percentage of MxM form as a function of the incubation
time at 4 °C(r)and37°C(d) is reported.
Fig. 5. Kinetic analysis of the M=M to MxM conversion for BS-RNase
(A), P19A-BS-RNase (B), L28Q-BS-RNase (C) and P19A/L28Q-BS-
RNase (D). The percentage of the MxM form as a function of the
incubation time at 4 °C(r)and37°C(d) is reported for each dimeric
protein.
4734 C. Ercole et al.(Eur. J. Biochem. 270) Ó FEBS 2003

BS-RNase. The former substitution destabilizes the mono-
meric form, and the latter specifically favours the swapped
dimer.
Acknowledgements
We thank the CIMCF of the University ÔFederico IIÕ of Naples where
we acquired some of the NMR spectra. We are very grateful to
Professor Giuseppe D’Alessio for critical reading of the manuscript. We
also thank Professor Alberto Di Donato and Dr Valeria Cafaro for
their kind hospitality and advice on protein expression and purification.
Financial support was from CNR (Agenzia 2000) and Ministero
dell’Istruzione e dell’Universita
`
(Prin 2002).
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Ó FEBS 2003 Structural properties of BS-RNase variants (Eur. J. Biochem. 270) 4735