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Analysis of the stability of the spermadhesin PSP-I ⁄ PSP-II heterodimer Effects of Zn2+ and acidic pH Marı́a Asunción Campanero-Rhodes1, Margarita Menéndez1, José Luis Sáiz1, Libia Sanz2, Juan José Calvete2 and Dolores Solı́s1 1 Instituto de Quı́mica Fı́sica ‘Rocasolano’, CSIC, Madrid, Spain 2 Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain Keywords heterodimer dissociation; PSP-I ⁄ PSP-II; spermadhesins; thermal stability; Zn2+ Correspondence D. Solı́s, Instituto de Quı́mica Fı́sica 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. Proteins are designed to have a particular activity in a specific environment, and their fold and assembly are intimately related to this physiological function. Information 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 stability [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 Abbreviations DSC, differential scanning calorimetry. FEBS Journal 272 (2005) 5663–5670 ª 2005 FEBS 5663 Stability of PSP-I ⁄ PSP-II heterodimer (0.3–0.7 mm) [3,4], reaching the spermatozoa at ejaculation [5]. Seminal plasma also contains a large number 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 spermatozoal 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 Å 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 contributes to maintaining sperm with high viability, motility, and mitochondrial activity [14]. In addition, PSP-I and PSP-II are immunostimulatory for lymphocyte activity in vitro [15]. Lymphocyte 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 migrationinducing activity of PSP-I ⁄ PSP-II, and possibly of the PSP-II subunit, is mediated by the stimulation of resident macrophages, which release a neutrophil chemotactic 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 subunits have been investigated thoroughly. The PSP-II subunit exhibits mannose 6-phosphate and heparin 5664 M. A. Campanero-Rhodes et al. 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 significance. In this context, it is noteworthy that the stimulatory activity of PSP-II on macrophages is selectively inhibited by mannose 6-phosphate [17]. Here we show that, in the presence of physiological concentrations of Zn2+, the stability of the heterodimer is significantly lowered, promoting at low protein 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 topology 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. PSPI ⁄ PSP-II thermal denaturation was irreversible [12], but the thermal denaturation profiles were practically scan-rate independent. Experimental curves were therefore phenomenologically analyzed using a sigmoidal function (see Experimental procedures) from which a T1 ⁄ 2 (temperature at which 50% of the protein is denatured) of 62.2
C can be estimated (Table 1). The far-UV and near-UV CD spectra of PSP-I ⁄ PSPII 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 temperature 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 ⁄ 2 falls FEBS Journal 272 (2005) 5663–5670 ª 2005 FEBS Stability of PSP-I ⁄ PSP-II heterodimer M. A. Campanero-Rhodes et al. Fig. 1. Near-UV CD of PSP-I ⁄ PSP-II. Variation with temperature (A) and effect of Zn2+ on the thermal denaturation (B) of the heterodimer. Spectra were obtained for 1 mgÆmL)1 PSP-I ⁄ PSP-II solutions in 20 mM Hepes, pH 7.0. (A) Representative spectra acquired at 25
C (h), 50
C (n), 56
C (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) mM Zn2+. 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 (T1 ⁄ 2) and DSC (Tm, DHcal). ND, Not determined. pH 7 3.8 Additive (mM) T1 ⁄ 2 (
C) Tm (
C) DHCAL (kJÆmol)1) None ZnCl2 (0.5) ZnCl2 (0.5) +EDTA (1) ZnCl2 (4) CaCl2 (5) None 62.2 ± 0.5 53.2 ± 0.2 ND 60.7 ± 0.3 59.8 ± 0.1 60.8 ± 0.1 405 ± 17 260 ± 20 440 ± 40 46.8 ± 0.2 ND ND 51.8 ± 0.3 61.6 ± 0.1 52.9 ± 0.6 240 ± 10 460 ± 30 330 ± 20 to 53.2
C, 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 cooperative unfolding unit. Thermal denaturation curves of PSP-I ⁄ PSP-II presented a single peak with a maximum at 60.5
C and an apparent enthalpy change of 439 kJÆ(mol dimer))1 [12]. We have since observed some differences among protein batches in the calorimetric enthalpy changes, with a mean ± SD DHcal of 405 ± 17 kJÆmol)1 (r, n ¼ 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 ± 0.3
C), thus serving as a useful gauge of the heterodimer thermostability. DSC data confirmed that, in the presence of ZnCl2, the thermal stability of PSP-I ⁄ PSP-II was substantially FEBS Journal 272 (2005) 5663–5670 ª 2005 FEBS reduced (Fig. 2A). As the Zn2+ concentration was increased, a concomitant decrease in both the transition temperature and the apparent enthalpy of denaturation was observed (Table 1), and, at 4 mm ZnCl2, protein precipitation occurred above 65
C. The destabilization induced by Zn2+ was reversed by the addition of EDTA to the sample (Fig. 2A). On the other hand, no significant decrease in the heterodimer stability 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 denaturation decreased
75 kJÆmol)1 and the transition temperature was 8
C 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-idealcomponent 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 molecular 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. 5665 Stability of PSP-I ⁄ PSP-II heterodimer M. A. Campanero-Rhodes et al. Fig. 3. Dependence on protein concentration of the gel filtration chromatographic behaviour of PSP-I ⁄ PSP-II. Effects of Zn2+ (B) and acidic pH (C). A 0.75 lgÆmL)1 solution of 125I-labelled PSP-I ⁄ PSP-II alone (dot lines) or in the presence of 5.5 mgÆmL)1 unlabelled PSPI ⁄ PSP-II (continuous lines) was chromatographed on a Superose 12 column equilibrated with 10 mM Tris ⁄ HCl (pH 7.8) ⁄ 0.15 M NaCl ⁄ 0.02% NaN3 (Tris ⁄ NaCl), in the absence (A) or presence of 2 mM ZnCl2 (Tris ⁄ NaCl-Zn2+) (B), or with 50 mM sodium acetate ⁄ acetic acid buffer (pH 4) ⁄ 0.15 M NaCl ⁄ 0.02% NaN3 (C). In (B), the elution profile of a 0.06 mgÆmL)1 solution of 125I-labelled PSPI ⁄ PSP-II in Tris ⁄ NaCl containing 2 mM Zn2+ is also shown (dashed line). Fig. 2. DSC profiles of the thermal denaturation of PSP-I ⁄ PSP-II. Effect of Zn2+ (A) and pH (B). The excess heat capacity function (DCp) of PSP-I ⁄ PSP-II was determined at a scanning rate of 20
CÆh)1 in 20 mM Hepes, pH 7 (thick solid line in A and B) or (A) in the same buffer containing 0.5 mM Zn2+ (thin solid line), 0.5 mM Zn2+ plus 1 mM EDTA (dash line), 1 mM Zn2+ (dash-dot line) or 4 mM Zn2+ (dot line) or (B) in 10 mM citric acid ⁄ sodium citrate, pH 3.8 (dot line). This behaviour was not related to the radioiodination of the protein because a 0.75 lgÆmL)1 solution of 125 I-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 associationdissociation equilibrium leading to dissociation of the heterodimer at protein concentrations in the low nanomolar range. The presence of 3 mm CaCl2 did not modify the chromatographic behaviour of PSP-I ⁄ PSP-II. In contrast, 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 radioiodinated 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 5666 chromatography, as shown in Fig. 4A for a 6 lgÆmL)1 solution of 125I-labelled PSP-I ⁄ PSP-II analysed immediately after the addition of 2 mm ZnCl2 or after an incubation period of either 2 h or 16 h. The composition of the fractions eluted from the column was analysed 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 PSPI ⁄ PSP-II was chromatographed under the above conditions, 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+ contained 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 analysis. 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 FEBS Journal 272 (2005) 5663–5670 ª 2005 FEBS M. A. Campanero-Rhodes et al. Fig. 4. Effect of incubation of PSP-I ⁄ PSP-II heterodimer with Zn2+ at low protein concentration. Gel filtration behaviour (A) and analysis 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)1 on a Superose 12 column equilibrated with Tris ⁄ NaCl-Zn2+ immediately after the addition of 2 mM ZnCl2 (continuous line) or after incubation for either 2 h (dash line) or 16 h (dot line) with the cation. Then 1-mL fractions were collected. The composition of selected fractions of 0 h (d, s) and 16 h (m, n) 125I-labelled PSP-I ⁄ PSP-II-Zn2 was subsequently analysed by RP-HPLC (B) on a C18 column eluted with an acetonitrile gradient (indicated by the line), as described in Experimental procedures. Control 125I-labelled PSP-I ⁄ PSP-II (h). from 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 of 125I-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 125 I-labelled PSP-I ⁄ PSP-II at pH 4 resulted in broadening 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 thermal 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 FEBS Journal 272 (2005) 5663–5670 ª 2005 FEBS Stability of PSP-I ⁄ PSP-II heterodimer the hydrophobic core of the CUB domain. This core is conserved in the X-ray structures of proteins containing the CUB signature, including the mannanbinding 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 tertiary 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 transition temperature of the thermal denaturation. This destabilization occurs with the dissociation of the heterodimer 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 seminal plasma definitely limits the level of free zinc available. The neutral to alkaline pH of normal boar seminal plasma also prevents dissociation of the PSPI ⁄ PSP-II heterodimer, and perhaps contributes to the reported protective action of this spermadhesin complex on sperm viability [14]. In fact, whereas free PSPI 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 quantitatively engaged in complex formation with PSP-I. Therefore, the heparin and mannose 6-phosphate binding 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 thermal stability of PSP-I ⁄ PSP-II and favours its dissociation at low protein concentrations. 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 intersubunit interactions may contribute critically to the thermal stability. The heterodimer interface is largely hydrophobic, consisting of a central, solvent-inaccessible hydrophobic core flanked at both sides by a cluster of polar ⁄ charged residues and a solvent-exposed aromatic amino acid (Fig. 5) [13]. In addition to 5667 Stability of PSP-I ⁄ PSP-II heterodimer M. A. Campanero-Rhodes et al. 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 artificial insemination. Experimental procedures Isolation and radioiodination of PSP-I ⁄ PSP-II Fig. 5. Ribbon diagram of the PSP-I ⁄ PSP-II heterodimer showing the characteristics of the dimer interface. Residues of the hydrophobic 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. The PSP-I ⁄ PSP-II heterodimer was isolated from the nonheparin-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 manufacturer’s recommendations. The radioiodinated protein was indistinguishable from the corresponding unlabelled one on SDS ⁄ PAGE and autoradiography. CD spectra hydrophobic contacts and van der Waals interactions, a salt bridge and a number of hydrogen bonds contribute to stabilization of the heterodimeric association. Weakening of these polar interactions, substantiated by the increased tendency of PSP-I ⁄ PSP-II to dissociate at low protein concentrations, because of protonation of the groups involved or as a result of Zn2+ complexation undoubtedly plays a part in the decrease in heterodimer thermal stability. For example, protonation and ⁄ or the potential involvement of Asp2 in Zn2+ coordination by PSP-I would prevent the formation 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 heterodimer, and the acidic environment of the cervical, uterine and intraluminal sperm reservoir fluids [18] may eventually contribute to pH-induced destabilization of the quaternary 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 consequence, 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 PSPII 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 5668 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 recorded 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 different scans were acquired and averaged for each sample. For all CD spectra, the corresponding buffer baseline was subtracted. 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 85
C at a heating rate of 0.33
CÆmin)1, allowing the temperature to equilibrate for 5 min before recording the spectrum. Variations in ellipticity were monitored every 0.2
C at 268 nm, and the complete spectrum was recorded every 5–15
C, after an equilibration time of 1–5 min at the selected temperature. No differences between the ellipticity values acquired at a given wavelength and those obtained from the spectra FEBS Journal 272 (2005) 5663–5670 ª 2005 FEBS Stability of PSP-I ⁄ PSP-II heterodimer M. A. Campanero-Rhodes et al. where T is the absolute temperature, T1 ⁄ 2 is the half transition temperature, R is the gas constant, A is the temperature constant accounting for the ratio between the native and denatured states, and QD(T) and QN(T) are the ellipticity of the denatured and native states at temperature T. QD and QN were approximated as linear functions of temperature [Qi(T) ¼ Qi(T0) + mi(T ) T0), where T0 is the reference temperature and mi is temperature dependence of Qi for i ¼ N or D]. belled PSP-I ⁄ PSP-II, and 500 lL of this mixture was analysed by RP-HPLC on a 5-lm Hypersil ODS C18 column (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 monitored 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. DSC Analytical ultracentrifugation For DSC, samples were dialyzed extensively against 20 mm Hepes buffer, pH 7, in the absence or presence of different concentrations of ZnCl2 or 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. Sedimentation equilibrium experiments were performed by centrifugation of 80-lL samples of concentration 0.5 mgÆmL)1, at 30 000 g and 20
C, 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]. were observed. Thermal denaturation profiles were described in terms of the following sigmoidal function: HðTÞ ¼ HD ðTÞ  ½HD ðTÞ  HN ðTÞ=f1  exp½AðT  T1=2 Þ= RTT1=2 g 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) NaN3 and, where stated, ZnCl2 or CaCl2 at the indicated concentration. Alternatively, 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 column, the injection syringe was previously blocked for 3 h at 20
C with 10% (v ⁄ v) Tween 20 (Sigma, St Louis, MO, USA). Then 1-mL fractions were collected into vapex sample 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 fractions was monitored by HPLC analysis, as described below. RP-HPLC Fractions collected from the gel filtration chromatography of 125I-labelled PSP-I ⁄ PSP-II were mixed with 250 lg unla- FEBS Journal 272 (2005) 5663–5670 ª 2005 FEBS Acknowledgements We thank DGICYT (BQU2000-1501-C02-02, BQU200303550-C03-03, BIO2003-01952 and BFU2004-1432) for financial support. We also thank Professor Heriberto Rodrı́guez-Martı́nez (Faculty of Veterinary Medicine, Clinical Centre Ultuna, Uppsala, Sweden) for critical reading of the manuscript and helpful discussions. References 1 Minton AP (2005) Influence of macromolecular crowding upon the stability and state of association of proteins: predictions and observations. J Pharm Sci 94, 1668–1675. 2 Mann T & Lutwak-Mann C (1981) Biochemistry of seminal plasma and male accessory fluids: application to andrological problems. 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