In boar seminal plasma, for example, the concentration of Zn2+ is surprisingly high Keywords heterodimer dissociation; PSP-I⁄ PSP-II; spermadhesins; thermal stability; Zn 2+ Corresponden
Trang 1Marı´a Asuncio´n Campanero-Rhodes1, Margarita Mene´ndez1, Jose´ Luis Sa´iz1, Libia Sanz2,
Juan Jose´ Calvete2and Dolores Solı´s1
1 Instituto de Quı´mica Fı´sica ‘Rocasolano’, CSIC, Madrid, Spain
2 Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain
Proteins are designed to have a particular activity in a
specific environment, and their fold and assembly are
intimately related to this physiological function
Infor-mation on the organization of the protein structure,
however, is usually acquired in simple buffer systems,
far removed from the complex conditions encountered
in intracellular and extracellular spaces and fluids
Besides the crucial influence of the local concentration
of macromolecules, the presence of co-solutes may
have a decisive effect on protein conformation and
sta-bility [1]
Seminal plasma is a composite fluid, comprising secretions from the testes, epididymis and accessory sex glands It is not merely a vehicle for the ejaculated sperm but it is also involved in numerous activities in the male and female reproductive tract, ensuring the viability and fertilizing capacity of spermatozoa The seminal plasma contains abundant concentrations of different amino acids, peptides, lipids, fatty acids and various osmolytes, and it is an important source
of cations [2] In boar seminal plasma, for example, the concentration of Zn2+ is surprisingly high
Keywords
heterodimer dissociation; PSP-I⁄ PSP-II;
spermadhesins; thermal stability; Zn 2+
Correspondence
D Solı´s, Instituto de Quı´mica Fı´sica
Rocasolano, Serrano 119, 28006 Madrid,
Spain
Fax: +34 91 564 24 31
Tel: +34 91 561 94 00
E-mail: d.solis@iqfr.csic.es
(Received 20 June 2005, revised 7 September
2005, accepted 14 September 2005)
doi:10.1111/j.1742-4658.2005.04974.x
Spermadhesins are a family of 12–16 kDa proteins with a single CUB domain PSP-I and PSP-II, the most abundant boar spermadhesins, are present in seminal plasma as a noncovalent heterodimer Dimerization markedly affects the binding ability of the subunits Notably, heparin and mannose 6-phosphate binding abilities of PSP-II are abolished, indicating that the corresponding binding sites may be located at (or near) the dimer interface Pursuing the hypothesis that cryptic binding sites in PSP-I⁄ PSP-II may be exposed in specific physiological environments, we examined the influence of Zn2+and acidic pH on the heterodimer stability According to near-UV CD spectra, the core native fold is preserved in the presence of physiological concentrations of Zn2+, a cation unusually abundant in boar seminal plasma However, the thermostability of the heterodimer decreases significantly, as observed by CD and differential scanning calorimetry The effect is Zn2+-specific and is reversed by EDTA Destabilization is also observed at acidic pH Gel filtration analysis using radioiodinated PSP-I⁄ PSP-II reveals that dissociation of the heterodimer at low (nanomolar) protein concentrations is promoted by both Zn2+and acidic pH Although the integrity of the heterodimer in seminal plasma seems to be guaranteed by its high concentration, dissociation may be facilitated in the female genital tract because of dilution of the protein in the intraluminal fluids of the cervix and the uterus, and the acidic fluid of the uterotubal junction Such a mechanism may be relevant in the regulation of uterine immune reactions
Abbreviations
DSC, differential scanning calorimetry.
Trang 2(0.3–0.7 mm) [3,4], reaching the spermatozoa at
ejac-ulation [5] Seminal plasma also contains a large
num-ber of different proteins that exert multiple effects on
sperm function, including a diversity of enzymes,
hormones, growth factors and transport proteins [6]
However, the precise role of most of the seminal
plasma proteins in sperm physiology remains obscure
Spermadhesins are a family of 12–16 kDa proteins
found in seminal plasma and⁄ or attached to the
sper-matozoal surface of a variety of mammalian species
(e.g boar, bull and horse) [7] These proteins are
composed of 109–133 amino acids, show a 40–60%
sequence identity, and contain a single CUB domain
[8] Members of the spermadhesin family have been
shown to bind zona pellucida glycoproteins, serine
proteinase inhibitors, phospholipids and⁄ or sulfated
glycosaminoglycans [9], suggesting that they may be
involved in different steps of the complex fertilization
process In the boar, spermadhesins represent about
75% of the total protein content of seminal plasma,
their concentration ranging from 0.6 to 7 mgÆmL)1
[10] PSP-I and PSP-II, the most abundant boar
spermadhesins, occur as a noncovalent heterodimer [11]
The secondary structure and stability of the PSP-I⁄
PSP-II heterodimer in solution has been investigated
[12], and the crystal structure solved at 2.4 A˚ resolution
[13] Both subunits consists of a compact ellipsoidal
b-sandwich structure organized into two five-stranded
(parallel and antiparallel) b-sheets
Accumulating evidence points to a role for PSP-I⁄
PSP-II as an exogenous modulator of both sperm
function and uterine immune activity, thus ensuring
reproductive success The PSP-I⁄ PSP-II complex
con-tributes to maintaining sperm with high viability,
motility, and mitochondrial activity [14] In addition,
PSP-I and PSP-II are immunostimulatory for
lympho-cyte activity in vitro [15] Lympholympho-cyte binding of PSP-I
has been demonstrated [16] Furthermore, the PSP-I⁄
PSP-II heterodimer and its isolated subunits induce the
recruitment of neutrophils into the peritoneal cavity
of rats [17] and pigs [18] The neutrophil
migration-inducing activity of PSP-I⁄ PSP-II, and possibly of the
PSP-II subunit, is mediated by the stimulation of
resi-dent macrophages, which release a neutrophil
chemo-tactic substance [19] In contrast, PSP-I appears to act
directly on neutrophils [17] The purpose of these
immunostimulatory activities would be to prevent
possible infections of the lower reproductive tract and
to provide a foreign-cell-free uterine environment for
the descending early embryos
The ligand-binding capabilities of the isolated
sub-units have been investigated thoroughly The PSP-II
subunit exhibits mannose 6-phosphate and heparin
binding abilities [20], whereas conflicting results on the heparin-binding ability of the PSP-I subunit have been reported [11,21,22] These binding sites are nonetheless cryptic in the heterodimer, which is typically isolated from the nonheparin-binding fraction of boar seminal plasma [11], raising the question of their biological sig-nificance In this context, it is noteworthy that the stimulatory activity of PSP-II on macrophages is selec-tively inhibited by mannose 6-phosphate [17]
Here we show that, in the presence of physiological concentrations of Zn2+, the stability of the hetero-dimer is significantly lowered, promoting at low pro-tein concentrations dissociation of the PSP-I and PSP-II subunits Similar behaviour is induced by acidic
pH The results point to the possibility that the cryptic binding sites in the PSP-I⁄ PSP-II heterodimer are exposed in the female genital tract environment
Results
CD spectroscopy The far-UV CD spectrum of PSP-I⁄ PSP-II exhibits a large positive band at 202 nm and a negative region
at 215 nm [12], as expected for the b-sandwich topol-ogy of the CUB domain [13] In addition, the near-UV
CD spectrum was dominated by the presence of a sharp positive band at 291 nm, in the tryptophan region (Fig 1A) Furthermore, the spectrum showed
a large negative region with minima around 287 and
268 nm Thermal denaturation of PSP-I⁄ PSP-II led to
a decrease in the intensity of both the positive and negative bands (Fig 1A) along with an increase in ellipticity below 250 nm These changes reflect the loss
of tertiary structure of the protein Monitoring of the decrease with temperature of the ellipticity at 268 nm facilitated tracing of the denaturalization process
PSP-I⁄ PSP-II thermal denaturation was irreversible [12], but the thermal denaturation profiles were practically scan-rate independent Experimental curves were there-fore phenomenologically analyzed using a sigmoidal function (see Experimental procedures) from which a
T1 ⁄ 2(temperature at which 50% of the protein is dena-tured) of 62.2C can be estimated (Table 1)
The far-UV and near-UV CD spectra of PSP-I⁄
PSP-II were not affected by the presence of ZnCl2 in the medium at concentrations up to 4 mm (data not shown) However, the stability of the heterodimer against thermal denaturation was significantly reduced,
as evidenced by monitoring the variation with tem-perature of the ellipticity at 268 nm (Fig 1B) At 0.5 mm ZnCl2, a concentration of Zn2+ in the range
of those reported for porcine seminal plasma, T1⁄ 2falls
Trang 3to 53.2C, and a further decrease was observed at
higher Zn2+concentrations (Table 1)
Differential scanning calorimetry (DSC)
In a former study [12], the thermal stability of the
PSP-I⁄ PSP-II heterodimer was analysed by DSC,
showing that the entire dimer constituted the
cooper-ative unfolding unit Thermal denaturation curves of
PSP-I⁄ PSP-II presented a single peak with a maximum
at 60.5C and an apparent enthalpy change of
439 kJÆ(mol dimer))1 [12] We have since observed
some differences among protein batches in the
calori-metric enthalpy changes, with a mean ± SD DHcal of
405 ± 17 kJÆmol)1 (r, n¼ 8) These variations are
not related to the protein concentration or the scan
rate used in the analysis However, the Tm values of
the DSC transitions were quite reproducible from
batch to batch (60.7 ± 0.3C), thus serving as a
use-ful gauge of the heterodimer thermostability
DSC data confirmed that, in the presence of ZnCl2,
the thermal stability of PSP-I⁄ PSP-II was substantially
reduced (Fig 2A) As the Zn2+ concentration was increased, a concomitant decrease in both the trans-ition temperature and the apparent enthalpy of dena-turation was observed (Table 1), and, at 4 mm ZnCl2, protein precipitation occurred above 65C The desta-bilization induced by Zn2+ was reversed by the addi-tion of EDTA to the sample (Fig 2A) On the other hand, no significant decrease in the heterodimer stabil-ity was observed in the presence of 4 mm CaCl2 (Table 1), emphasizing the specificity of the effect of
Zn2+ Thermal destabilization of PSP-I⁄ PSP-II was also noticed at acidic pH (Fig 2B) in the absence of Zn2+ cations At pH 3.8 the apparent enthalpy of denatura-tion decreased 75 kJÆmol)1 and the transition tem-perature was 8C lower (Table 1)
Ultracentrifugation and chromatographic behaviour
The sedimentation equilibrium data for PSP-I⁄ PSP-II (0.25–0.5 mgÆmL)1) could be fitted to a single-ideal-component model with a weight-average molecular mass of 27 933 Da, confirming that PSP-I⁄ PSP-II behaved in solution as a dimer No influence of Zn2+
at concentrations up to 4 mm on the average mole-cular mass of PSP-I⁄ PSP-II was observed at this protein concentration range
On gel filtration chromatography, the elution time
of PSP-I⁄ PSP-II at concentrations of, or above, 0.01 mgÆmL)1 was 26 min, consistent with the time predicted for the dimer However, analysis of the gel filtration behaviour using 125I-labelled PSP-I⁄ PSP-II revealed a broadening of the peak at lower protein concentrations (Fig 3A), with the appearance of minor species at the elution volume of the isolated subunits
Fig 1 Near-UV CD of PSP-I ⁄ PSP-II Variation with temperature (A) and effect of Zn 2+ on the thermal denaturation (B) of the heterodimer Spectra were obtained for 1 mgÆmL)1PSP-I ⁄ PSP-II solutions in 20 m M Hepes, pH 7.0 (A) Representative spectra acquired at 25 C (h), 50 C
(n), 56C (n), 62 C (m), 70 C (s) and 77 C (d) C (B) Variation in ellipticity at 268 nm with temperature monitored in the absence (s) or in the presence of 0.5 (n) or 4 (h) m M Zn 2+ The continuous lines correspond to the fit of the experimental data to a sigmoidal function.
Table 1 Thermodynamic parameters of the thermal denaturation
of PSP-I ⁄ PSP-II as determined by CD (T 1 ⁄ 2 ) and DSC (Tm, DHcal).
ND, Not determined.
pH
Additive
(m M )
T 1⁄ 2
(C)
T m
(C)
DH CAL
(kJÆmol)1)
7 None 62.2 ± 0.5 60.7 ± 0.3 405 ± 17
ZnCl 2 (0.5) 53.2 ± 0.2 59.8 ± 0.1 260 ± 20
ZnCl2(0.5)
+EDTA (1)
ND 60.8 ± 0.1 440 ± 40 ZnCl 2 (4) 46.8 ± 0.2 51.8 ± 0.3 240 ± 10
CaCl2(5) ND 61.6 ± 0.1 460 ± 30
3.8 None ND 52.9 ± 0.6 330 ± 20
Trang 4This behaviour was not related to the radioiodination
of the protein because a 0.75 lgÆmL)1 solution of
125I-labelled PSP-I⁄ PSP-II was eluted as a single sharp
peak at 26 min when it was chromatographed in the
presence of unlabelled protein (Fig 3A) In contrast,
the results suggested the existence of an
association-dissociation equilibrium leading to association-dissociation of the
heterodimer at protein concentrations in the low
nano-molar range
The presence of 3 mm CaCl2 did not modify the
chromatographic behaviour of PSP-I⁄ PSP-II In
con-trast, the addition of 2 mm Zn2+ intensified the
deviation of the elution profile at low protein
concentrations from that of the dimer Thus, at PSP-I⁄
PSP-II concentrations below 0.06 mgÆmL)1, the
radio-iodinated protein was eluted as a broadened peak,
with a displacement of the maximum towards longer
elution times and a decrease in the total area of the
peak (Fig 3B) At a given protein concentration,
the changes in the profile became more intense when
the sample was preincubated with Zn2+ before the
chromatography, as shown in Fig 4A for a 6 lgÆmL)1 solution of 125I-labelled PSP-I⁄ PSP-II analysed imme-diately after the addition of 2 mm ZnCl2 or after an incubation period of either 2 h or 16 h The composi-tion of the fraccomposi-tions eluted from the column was ana-lysed by RP-HPLC, using a protocol designed for the separation of the PSP-I and PSP-II subunits [11] When a mixture of unlabelled and 125I-labelled
PSP-I⁄ PSP-II was chromatographed under the above condi-tions, two radioactivity peaks were co-eluted with the unlabelled PSP-I and PSP-II subunits, together with a third radioactive peak, appearing at the void volume, which corresponded to free 125I (Fig 4B) A similar analysis of the material eluted from the gel filtration column revealed that the first fractions of the sample eluted immediately after the addition of Zn2+ con-tained both PSP-I and PSP-II subunits, whereas the fractions eluted later were mainly composed of PSP-II, supporting the dissociation of the heterodimer (Fig 4B) Preincubation of the 125I-labelled PSP-I⁄ PSP-II sample with Zn2+ resulted in a gradual decrease in the amount of PSP-I eluted from the gel filtration column, so that, after incubation for 16 h, only the PSP-II subunit was detected by HPLC analy-sis The 125I-labelled PSP-I subunit became partially adsorbed to the vials used for preincubation, as revealed by radioactivity monitoring and SDS⁄ PAGE followed by autoradiography of the material eluted
Fig 2 DSC profiles of the thermal denaturation of PSP-I ⁄ PSP-II.
Effect of Zn 2+ (A) and pH (B) The excess heat capacity function
(DCp) of PSP-I ⁄ PSP-II was determined at a scanning rate of
20 CÆh)1in 20 m M Hepes, pH 7 (thick solid line in A and B) or (A)
in the same buffer containing 0.5 m M Zn 2+ (thin solid line), 0.5 m M
Zn 2+ plus 1 m M EDTA (dash line), 1 m M Zn 2+ (dash-dot line) or
4 m M Zn 2+ (dot line) or (B) in 10 m M citric acid ⁄ sodium citrate,
pH 3.8 (dot line).
Fig 3 Dependence on protein concentration of the gel filtration chromatographic behaviour of PSP-I ⁄ PSP-II Effects of Zn 2+
(B) and acidic pH (C) A 0.75 lgÆmL)1solution of 125 I-labelled PSP-I ⁄ PSP-II alone (dot lines) or in the presence of 5.5 mgÆmL)1unlabelled
PSP-I ⁄ PSP-II (continuous lines) was chromatographed on a Superose
12 column equilibrated with 10 m M Tris ⁄ HCl (pH 7.8) ⁄ 0.15 M
NaCl ⁄ 0.02% NaN 3 (Tris ⁄ NaCl), in the absence (A) or presence of
2 m M ZnCl 2 (Tris ⁄ NaCl-Zn 2+ ) (B), or with 50 m M sodium acet-ate ⁄ acetic acid buffer (pH 4) ⁄ 0.15 M NaCl ⁄ 0.02% NaN 3 (C) In (B), the elution profile of a 0.06 mgÆmL)1solution of 125 I-labelled
PSP-I ⁄ PSP-II in Tris ⁄ NaCl containing 2 m M Zn 2+ is also shown (dashed line).
Trang 5from the vial with SDS⁄ PAGE sample buffer The
remaining 125I-labelled PSP-I was nonspecifically
retained on the FPLC column (results not shown)
Overall, the results show Zn2+-enhanced dissociation
of the PSP-I and PSP-II subunits at low heterodimer
concentrations No enhancing effect of Mg2+ on the
dissociation of125I-labelled PSP-I⁄ PSP-II samples was
observed at concentrations up to 30 mm
The heterodimer dissociation was also enhanced at
acidic pH Gel filtration of a 0.75 lgÆmL)1 solution of
125I-labelled PSP-I⁄ PSP-II at pH 4 resulted in
broaden-ing of the peak and the appearance of species at the
elution volume of the isolated subunits (Fig 3C) The
addition of Zn2+ at this pH did not induce additional
changes in the chromatographic behaviour
Discussion
The near-UV CD spectrum of PSP-I⁄ PSP-II reflects
the specific environment of chiral aromatic side chains
in the tertiary structure of the folded protein, and the
band intensities decrease in a sigmoidal way as
ther-mal denaturation occurs In particular, the spectrum
is characterized by the presence of a sharp positive
band in the tryptophan absorption region (Fig 1A)
Both PSP-I and PSP-II subunits contain a single
tryptophan residue, which is accommodated within
the hydrophobic core of the CUB domain This core
is conserved in the X-ray structures of proteins con-taining the CUB signature, including the mannan-binding lectin-associated protease-2 (MASP-2) [23], its alternative splicing product Map19 [24], and the C1s protease of the C1 complex of complement [25] Thus, the Trp band can be regarded as a characteristic fingerprint of the native fold of PSP-I and PSP-II The near-UV CD spectra of the isolated PSP-I and PSP-II subunits are also characterized by the presence
of this band (data not shown), strongly suggesting that they preserve the overall fold of the CUB domain
In the presence of Zn2+ concentrations resembling physiological total amounts in seminal plasma, the ter-tiary structure of native PSP-I⁄ PSP-II is preserved However, the thermal stability of the heterodimer is significantly lower than in the absence of this cation,
as evidenced by a lower apparent enthalpy and trans-ition temperature of the thermal denaturation This destabilization occurs with the dissociation of the het-erodimer at low protein concentrations Nevertheless, the concentration of PSP-I⁄ PSP-II in seminal plasma is clearly high enough to guarantee the integrity of the dimer In addition, it should not be overlooked that complexation by other Zn2+-binding molecules in sem-inal plasma definitely limits the level of free zinc avail-able The neutral to alkaline pH of normal boar seminal plasma also prevents dissociation of the
PSP-I⁄ PSP-II heterodimer, and perhaps contributes to the reported protective action of this spermadhesin com-plex on sperm viability [14] In fact, whereas free
PSP-I has also been found in the heparin-binding fraction
of boar seminal plasma [26], no free PSP-II has been detected, indicating that PSP-I is synthesized in excess over PSP-II, and that the PSP-II subunit is quantita-tively engaged in complex formation with PSP-I Therefore, the heparin and mannose 6-phosphate bind-ing sites of PSP-II, which have been proposed to be located at the heterodimer interface [20], may not be exposed in the male genital tract
On the other hand, an acidic pH, such as that found
in seminal vesicle dysfunction, may decrease the ther-mal stability of PSP-I⁄ PSP-II and favours its dissoci-ation at low protein concentrdissoci-ations Previous DSC studies on the thermal denaturation of PSP-I⁄ PSP-II [12] showed that the whole dimer constituted the cooperative unfolding unit, suggesting that inter-subunit interactions may contribute critically to the thermal stability The heterodimer interface is largely hydrophobic, consisting of a central, solvent-inacces-sible hydrophobic core flanked at both sides by a clus-ter of polar⁄ charged residues and a solvent-exposed aromatic amino acid (Fig 5) [13] In addition to
Fig 4 Effect of incubation of PSP-I ⁄ PSP-II heterodimer with Zn 2+
at low protein concentration Gel filtration behaviour (A) and
analy-sis by RP-HPLC (B) of the composition of the fractions derived
from the gel filtration column (A) A 6 lgÆmL)1 solution of
125 I-labelled PSP-I ⁄ PSP-II was chromatographed at 0.5 mLÆmin)1on
a Superose 12 column equilibrated with Tris ⁄ NaCl-Zn 2+ immediately
after the addition of 2 m M ZnCl 2 (continuous line) or after
incuba-tion for either 2 h (dash line) or 16 h (dot line) with the caincuba-tion Then
1-mL fractions were collected The composition of selected
frac-tions of 0 h (d, s) and 16 h (m, n)125I-labelled PSP-I ⁄ PSP-II-Zn 2
was subsequently analysed by RP-HPLC (B) on a C18column eluted
with an acetonitrile gradient (indicated by the line), as described in
Experimental procedures Control 125 I-labelled PSP-I ⁄ PSP-II (h).
Trang 6hydrophobic contacts and van der Waals interactions,
a salt bridge and a number of hydrogen bonds
contrib-ute to stabilization of the heterodimeric association
Weakening of these polar interactions, substantiated
by the increased tendency of PSP-I⁄ PSP-II to
dissoci-ate at low protein concentrations, because of
protona-tion of the groups involved or as a result of Zn2+
complexation undoubtedly plays a part in the decrease
in heterodimer thermal stability For example,
proto-nation and⁄ or the potential involvement of Asp2 in
Zn2+coordination by PSP-I would prevent the
forma-tion of two strong hydrogen bonds with residues
Tyr108 and Ser110 from PSP-II [13]
The entry of semen into the female genital tract is
associated with dilution of the PSP-I⁄ PSP-II
heterodi-mer, and the acidic environment of the cervical, uterine
and intraluminal sperm reservoir fluids [18] may
eventu-ally contribute to pH-induced destabilization of the
qua-ternary structure of the spermadhesin complex These
changes, possibly in conjunction with other factors or
conditions encountered in the female tract, may give rise
to separation of the PSP-I⁄ PSP-II subunits As a
conse-quence, the heparin and mannose 6-phosphate binding
sites on PSP-II would be exposed It is important to
emphasize that the reported stimulatory activity of
PSP-II on macrophages is selectively inhibited by mannose
6-phosphate [17], suggesting the involvement of this
binding site in the proposed activity of PSP-II as a
post-mating inflammation mediator The neutrophil recruitment induced by PSP-I appears to use a different mechanism, acting directly on neutrophils [17] Thus, the dissociation of the PSP-I⁄ PSP-II heterodimer in the female genital tract may be of physiological significance
It may be of relevance for the regulation of the duration and magnitude of uterine immune reactions, particularly
in the search of strategies to optimize fecundity in artifi-cial insemination
Experimental procedures
Isolation and radioiodination of PSP-I⁄ PSP-II The PSP-I⁄ PSP-II heterodimer was isolated from the non-heparin-binding fraction of boar seminal plasma by gel filtration chromatography as described [11] The protein (300 lg) was labelled with 0.2 mCi 125I using Iodogen (Pierce, Rockford, IL, USA), according to the manufac-turer’s recommendations The radioiodinated protein was indistinguishable from the corresponding unlabelled one on SDS⁄ PAGE and autoradiography
CD spectra PSP-I⁄ PSP-II samples were dialyzed extensively against
20 mm Hepes buffer, pH 7, in the absence or presence of different concentrations of ZnCl2 CD spectra were recor-ded in a JASCO J-720 spectropolarimeter (Jasco Corp., Tokyo, Japan), fitted with a water bath thermostatted cell holder, or in a J-810 spectropolarimeter, equipped with a peltier temperature control system, using a band width of 0.2 nm and a response time of 2 s Far-UV spectra were recorded in 0.02 and 0.1 cm pathlength quartz cells at a protein concentration of 1 and 0.2 mgÆmL)1, respectively Near-UV spectra were acquired at 1.0 mgÆmL)1 protein concentration in 1 cm pathlength cells At least three differ-ent scans were acquired and averaged for each sample For all CD spectra, the corresponding buffer baseline was sub-tracted The observed ellipticities were converted into mean residue ellipticities using a mean molecular mass per residue
of 127.4 This value was calculated by dividing the average molecular mass obtained by MALDI MS (28 664 Da) by the number of amino-acid residues of the mature protein sequence (225 residues)
Thermal denaturation experiments were carried out by increasing the temperature from 15 to 85C at a heating rate of 0.33CÆmin)1, allowing the temperature to equili-brate for 5 min before recording the spectrum Variations
in ellipticity were monitored every 0.2C at 268 nm, and the complete spectrum was recorded every 5–15C, after
an equilibration time of 1–5 min at the selected tempera-ture No differences between the ellipticity values acquired
at a given wavelength and those obtained from the spectra
Fig 5 Ribbon diagram of the PSP-I ⁄ PSP-II heterodimer showing
the characteristics of the dimer interface Residues of the
hydro-phobic core are coloured in yellow, and hydrogen bonds formed at
both sides by main-chain or side-chain atoms (coloured in CPK) of
flanking polar residues are represented by dotted lines The lateral
chains of PSP-I Glu101 and PSP-II Arg43, which are involved in a
salt bridge, are also shown Residues are numbered according to
the amino-acid sequence of the mature protein In the lower part of
the figure, PSP-I Asp2, a potential zinc ligand, forms two strong
hydrogen bonds with residues Tyr108 and Ser110 from PSP-II.
Trang 7were observed Thermal denaturation profiles were
des-cribed in terms of the following sigmoidal function:
HðTÞ ¼ HDðTÞ ½HDðTÞ HNðTÞ=f1 exp½AðT T1=2Þ=
RTT1=2g
where T is the absolute temperature, T1⁄ 2is the half transition
temperature, R is the gas constant, A is the temperature
con-stant accounting for the ratio between the native and
dena-tured states, and QD(T) and QN(T) are the ellipticity of the
denatured and native states at temperature T.QDandQNwere
approximated as linear functions of temperature [Qi(T)¼
Qi(T0) + mi(T) T0), where T0is the reference temperature
and miis temperature dependence ofQifor i¼ N or D]
DSC
For DSC, samples were dialyzed extensively against 20 mm
Hepes buffer, pH 7, in the absence or presence of different
concentrations of ZnCl2or CaCl2, unless otherwise stated
DSC measurements were performed using a Microcal MCS
instrument (Microcal, Inc., Northampton, MA, USA) at a
heating rate of 0.33 KÆmin)1 and under an extra constant
pressure of 2 atm The standard Microcal origin software
was used for data acquisition and analysis The excess heat
capacity functions were obtained after subtraction of the
buffer baseline Reversibility of the transitions was checked
by performing a second analysis after the first scan
Gel filtration chromatography
Gel filtration was carried out on a Superose 12 HR 10⁄ 30
column (Pharmacia LKB Biotechnology, Uppsala, Sweden)
equilibrated with 10 mm Tris⁄ HCl (pH 7.8) ⁄ 0.15 m NaCl
(Tris⁄ NaCl), containing 0.02% (w ⁄ v) NaN3and, where
sta-ted, ZnCl2 or CaCl2 at the indicated concentration
Alter-natively, the column was equilibrated with 50 mm sodium
acetate⁄ acetic acid buffer (pH 4)⁄ 0.15 m NaCl⁄ 0.02%
(w⁄ v) NaN3 The flow rate was 0.5 mLÆmin)1, and the
elution was monitored at 280 nm Control proteins were
chromatographed under similar conditions
For loading radioiodinated PSP-I⁄ PSP-II on to the
col-umn, the injection syringe was previously blocked for 3 h at
20C with 10% (v ⁄ v) Tween 20 (Sigma, St Louis, MO,
USA) Then 1-mL fractions were collected into vapex
sam-ple tubes (PerkinElmer, Turku, Finland), similarly blocked
with 0.5% (v⁄ v) Tween 20 for 16 h at 20 C, and their
radioactivity was measured in an LKB MiniGamma counter
(LKB Wallac, Turku, Finland) Composition of the
frac-tions was monitored by HPLC analysis, as described below
RP-HPLC
Fractions collected from the gel filtration chromatography
of125I-labelled PSP-I⁄ PSP-II were mixed with 250 lg
unla-belled PSP-I⁄ PSP-II, and 500 lL of this mixture was ana-lysed by RP-HPLC on a 5-lm Hypersil ODS C18column (Sugelabor, Madrid, Spain), eluted at 1 mLÆmin)1 with an acetonitrile gradient in 0.1% (v⁄ v) trifluoroacetic acid as follows: (a) 35% acetonitrile isocratically for 5 min; (b) 35–40% (v⁄ v) for 5 min; (c) 40–50% for 80 min; (d) 50–70% (v⁄ v) acetonitrile for 10 min The column was re-equilibrated with 35% (v⁄ v) acetonitrile for 20 min before application of a new sample The elution was moni-tored at 280 nm, and 3 mL fractions were collected The elution position of the radioiodinated PSP-I and PSP-II subunits was checked by analysing control 125I-labelled PSP-I⁄ PSP-II under the same conditions
Analytical ultracentrifugation Sedimentation equilibrium experiments were performed
by centrifugation of 80-lL samples of concentration 0.5 mgÆmL)1, at 30 000 g and 20C, in an Optima XL-A analytical ultracentrifuge (Beckman Coulter Instruments, Inc., Richmond, CA, USA) equipped with UV-Vis optics and An50Ti analytical rotor Data were collected using
12 mm pathlength double-sector six-channel centre pieces with quartz windows Under these conditions, equilibrium was reached before 12 h of centrifugation Baseline offsets were determined from radial scans of the samples run for
6 h at 160 000 g Weight-average molecular masses, Mw, were calculated with the xlaeq program, using the signal conservation algorithm [27]
Acknowledgements
We thank DGICYT (BQU2000-1501-C02-02, BQU2003-03550-C03-03, BIO2003-01952 and BFU2004-1432) for financial support We also thank Professor Heriberto Rodrı´guez-Martı´nez (Faculty of Veterinary Medicine, Clinical Centre Ultuna, Uppsala, Sweden) for critical reading of the manuscript and helpful discussions
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