Báo cáo khoa học: High-pressure effects on horse heart metmyoglobin studied by small-angle neutron scattering pdf

High-pressure effects on horse heart metmyoglobin studied by small-angle neutron scattering Camille Loupiac 1 , Marco Bonetti 2 , Serge Pin 3 and Patrick Calmettes 1 1 Laboratoire Le ´ on Brillouin, UMR 12 CNRS, 2 Service de Physique de l’Etat Condense ´ , and 3 Service de Chimie Mole ´ culaire, URA 331 CNRS, DSM/DRECAM, CEA de Saclay, Gif-sur-Yvette, France Small-angle neutron scattering experiments were performed on horse azidometmyoglobin (MbN 3 ) at pressures up to 300 MPa. Other spectroscopic techniques have shown that a reorganization of the secondary structure and of the active site occur in this pressure range. The present measurements, performed using various concentrations of MbN 3 , show that the compactness of the protein is not altered as the value of its radius of gyration remains constant up to 300 MPa. The value of the second virial coefficient of the protein solution indicates that the interactions between the molecules are always strongly repulsive even if their magnitude decreases with increasing pressure. Taking advantage of the pressure- induced contrast variation, these experiments allow the partial specific volume of MbN 3 to be determined as a function of pressure. Its value decreases by 5.4% between atmospheric pressure and 300 MPa. In this pressure range the isothermal compressibility of hydrated MbN 3 is found to be almost constant. Its value is (1.6 ± 0.1) 10 )4 MPa )1 . Keywords: myoglobin; pressure; SANS; partial volume; compressibility. The structure of proteins and their solvent interactions can be modified by temperature, pH or chemicals. The appli- cation of hydrostatic pressure to a protein solution also provides a manner to alter these physical properties [1–4]. The stability of proteins in very different extreme environ- mental conditions is of great importance for many biotech- nological applications, notably food processing. Therefore, the various states that proteins can adopt under pressure is a matter of growing interest. In general, protein–ligand binding is affected by pressures lower than 400 MPa. Furthermore, protein denaturation and unfolding may occur at higher pressures [5–8]. Studies of protein stability by means of various spectroscopic techniques have shown that increasing pressure reduces the partial volume of the molecule through compression and conformational changes. Although matter is always compressible, electrostriction of charged and polar side chains, hydrophobic hydration, hydrogen bonds stabilization and the elimination of packing defects are considered to be the main causes for this volume change [9–15]. The effects of pressure on hemeproteins have been the subject of numerous investigations. Optical absorption [16– 21], fluorescence [22], FTIR [23–25], Raman [26], and NMR [27–29] spectroscopies, and laser flash photolysis [30–32] have all shown that pressures near 300 MPa leads to subtle local rearrangements of the protein structure and that some intermediate states preceding unfolding probably appear. Therefore, it is important to determine whether the modi- fications observed at the level of the active site of myoglobin [17,18,20,21,26–29] and the reorganization of the secondary structure with an alteration of the electrostatic and hydro- gen-bond array [23,24] are related to a change in the tertiary structure of the protein. In order to reply these questions we report here for the first time, the results of small-angle neutron scattering (SANS) experiments performed on myoglobin (Mb) under pressure. Quite generally, SANS can provide information about the partial volume of proteins, their interactions, their size and their conformation [33]. The scattering measure- ments were carried out in heavy water ( 2 H 2 O) at varying pressures up to 300 MPa as a function of protein concen- tration in order to determine the magnitude of the solute interactions and to allow for the elimination of their contribution to the forward scattered intensity and the apparent radius of gyration. Azidometmyoglobin (MbN 3 ) has a high stability. It was chosen for this study in order to avoid a mixture of aquo and hydroxy derivatives in metmyoglobin solutions or a contamination of either oxygen or carbon monoxide saturated myoglobin solutions by oxidized forms which could form under pressure [34]. MATERIALS AND METHODS Protein sample preparation High purity horse-heart Mb was purchased from Sigma. The lyophilized protein was first dissolved in water (H 2 O) and dialysed three times against H 2 O during 24 h to remove all the salts. The protein was then extensively dialysed (three dialyses of 24 h) against 2 H 2 O to achieve a complete exchange of the labile hydrogen atoms. For the SANS experiments a 0.1- M Bistris p 2 H 6.6 deuterated buffer was used so as to allow the highest contrast between the protein Correspondence to P. Calmettes, Laboratoire Le ´ on Brillouin, C.E.A. de Saclay, 91191 Gif-sur-Yvette, cedex, France. Fax: + 33 16908 8261, Tel.: + 33 16908 6476, E-mail: calmet@llb.saclay.cea.fr Abbreviations: SANS, small-angle neutron scattering; Mb, myoglobin; MbN 3 , azidometmyoglobin. (Received 26 February 2002, revised 10 June 2002, accepted 22 July 2002) Eur. J. Biochem. 269, 4731–4737 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03126.x and the solvent whilst also minimizing incoherent scattering from the hydrogen atoms. Bistris was chosen because its ionization constant should not be altered by pressure [35]. Sodium azide (NaN 3 ) was added to the aquometmyoglobin solution one day prior to the SANS experiments to ensure that the protein was almost fully liganded with N 3 [36]. This was checked by absorbance measurements in the visible region. The p 2 H of the solution was measured after ligandation and adjusted to 6.6 if necessary. The mother MbN 3 solution at about 60 mgÆcm )3 and the samples were preparedandstoredat4 °C. All samples were centrifuged at 20 000 g during 5 min at 15 °C prior to the SANS experiments. High-pressure cell During the SANS experiments the protein solutions were contained in a high-pressure cell made of stainless steel with two parallel thick sapphire windows. The optical path length was 5 mm and the maximum forward scattering angle h max ¼ 15°. A separator between the pressurizing fluid and the sample prevented the latter from contamina- tion. A hand driven pressure generator allowed the pressure to be gradually increased up to any value lower than about 300 MPa. No significant temperature increase was observed during pressurization performed at a rate of about 100 barÆmin )1 . Pressure was measured with an accuracy of ±0.3 MPa. SANS experiments The SANS experiments were performed with the PACE spectrometer at the Laboratoire Le ´ on Brillouin, Saclay, France. The neutron wavelength was k ¼ 1.1 nm. This gives access to wavenumber transfers, q, ranging from 0.07 to 0.75 nm )1 . q ¼ (4p/k)sin(h/2), where h is the scattering angle. The SANS spectra were collected at room temper- ature, near 20 °C. Each raw spectrum was divided by the corresponding transmission measured with a suitably attenuated beam after the removal of the beam stop located in front of the centre of the detector. The dialysis buffer and the empty cell scattering contributions were measured in the same conditions and subtracted from the spectrum of each protein sample. Finally, the results were corrected for the nonuniformity of the detector response by normalization to the incoherent scattering of a 1.00 mm path-length water sample. To check that protein aggregation did not occur during the course of the measurements, one hour spectra were recorded successively. The number of spectra was chosen according to the protein concentration so as to ensure the same statistical accuracy for all measurements after averag- ing. No protein aggregation was observed during the SANS experiments. Data analysis With respect to the solvent alone, the excess neutron intensity scattered forward from a protein solution is [33] Iðq ¼ 0; P; cÞ¼k B TcðPÞ½KðPÞ 2 oPðP; cÞ oc     À1 T;P ð1Þ where k B is Bolztmann’s constant, T the temperature (K) and P the pressure (MPa). c ¼ c(P) is the protein concen- tration (gÆcm )3 )andP(P,c) the osmotic pressure (MPa), both at pressure P. KðPÞ¼ b p N A M p À q 0 b ðPÞv p ðPÞ ! ð2Þ is the average specific contrast of the protein molecule with respect to the solvent. b p is the coherent scattering length (cm) of a molecule and M p its molar mass (gÆmol )1 ). M p @ 17.4 · 10 3 gÆmol )1 for Mb in 2 H 2 O [37]. N A is Avogadro’s number. v p (P) is the partial specific volume (cm 3 Æg )1 ) of the protein at pressure P. q¢ b (P)isthe scattering-length density (cm )2 ) of the buffer at the same pressure. As the salt concentration of the buffer is low it can be regarded as pure 2 H 2 O. Therefore its scattering length density is q 0 b ðPÞ¼b2 H 2 O q 2 H 2 O ðPÞ N A M 2 H 2 O ð3Þ where b 2 H 2 O is the coherent scattering length of a 2 H 2 O molecule. q 2 H 2 O (P)andM 2 H 2 O are the density (gÆcm )3 ) andthemolarmass(gÆmol )1 )of 2 H 2 O, respectively. At 20 °C, the pressure dependence of q 2 H 2 O (P) has only been measured up to 100 MPa [38]. Therefore, the left hand side of Eqn. 3 was calculated using the values of the density of H 2 O as a function of pressure [39] assuming that the molarities, q/M,of 2 H 2 OandH 2 Oare identical at 20 °C for pressures lower than 300 MPa. Up to 100 MPa this approximation leads to negligible errors compared to those resulting from small levels of hydro- gen contamination which always occur during sample preparation. As a first approximation, the partial specific volume of Mb was assumed to be independent of pressure and to have the value v p (0.1) ¼ 0.741 cm 3 Æg )1 [40] at atmospheric pres- sure P @ 0.1 MPa. Accordingly, in the following analysis of the scattering data the actual protein contrast given by Eqn. 2 has been replaced by the quantity K 0 ðPÞ¼ b p N A M p À q 0 b ðPÞv p ð0:1Þ ! ð4Þ where q 0 b ðP Þ is given by Eqn. 3. Using this expression and the virial expansion for the osmotic pressure, Eqn. 1 can be rewritten as follows cðPÞ½K 0 ðPÞ 2 Ið0;P;cÞ ¼ N A M p K 0 ðPÞ KðPÞ ! 2 1 þ 2A 2 ðPÞM p cðPÞþÁÁÁ Âà ð5Þ where A 2 (P) is the second virial coefficient of the solution. It describes the interactions between pairs of solute molecules and provides an estimate of the nonideality of the solution. A 2 (P) > 0 for repulsive interactions between the solute molecules. For relatively low solute concentrations, higher order terms in c(P) are negligible. As both K 0 (P)andK(P) do not depend on c(P), a plot of c(P)[K 0 (P)] 2 /I(0,P,c)vs.c(P) allows the value of A 2 (P)to be determined. The pressure dependence of the protein concentration was calculated by means of the expres- sion cðP Þ¼cð0:1Þq 2 H 2 O ðP Þ=q 2 H 2 O ð0:1Þ,wherec(0.1) and 4732 C. Loupiac et al. (Eur. J. Biochem. 269) Ó FEBS 2002 q 2 H 2 O ð0:1Þ are the protein concentration and the density of heavy water at atmospheric pressure, respectively. As in Eqn. 3, the values of c(P) were calculated using the densities of H 2 O. The SANS spectra from the protein were described by the Guinier approximation [41] Iðq; P; cÞffiIð0; P; cÞ exp½Àq 2 R 2 g ðP; cÞ=3ð6Þ where R g (P,c) is the apparent value (nm) of the radius of gyration of the protein at pressure P and concentration c(P). For an almost spherical solute particle this approximation is valid to within 1% for qR g (P,c) £ 1.3 [41]. For a Mb molecule, this corresponds to q £ 0.9 nm )1 .AlltheSANS spectra were collected within this range. The concentration dependence of the radius of gyration can be accounted for by ½R g ðP;cÞ À2 ¼½R g ðP; 0Þ À2 ½1 þ 2B 2 ðPÞM p cðPÞþÁÁÁ ð7Þ where R g (P,0) is the actual value of the radius of gyration of the protein and B 2 (P) a constant similar to A 2 (P)inEqn.5 but with a different value. For each pressure, R g (P,0) can be inferred from the intercept of the plot of [R g (P,c)] )2 as a function of c(P). RESULTS SANS measurements were performed at three protein concentrations measured at atmospheric pressure: 5.7, 11.7, and 16.2 mgÆcm )3 . Figure 1 shows the neutron scattering spectra obtained at 54, 154, and 302 MPa for thesampleat11.7mgÆcm )3 . For the spectrometer config- uration used in these experiments, the first two points at the lowest q-values are affected by a small contribution from the direct neutron beam. Consequently, no significant increase of the scattered intensity is observed for the smallest q-values. This demonstrates that no protein aggre- gation or oligomerization occurred in the samples, irrespec- tive of the pressure. To determine the apparent value of the radius of gyration, R g (P,c), of the MbN 3 molecule and the forward scattered intensity, I(0,P,c), Eqn 6 was fitted to these spectra and those from the samples at the other two concentrations. As shown in Fig. 2, the values of the actual radius of gyration, R g (P,0), at each pressure were inferred from R g (P,c) by extrapolation to c(P) ¼ 0 according to Eqn 7. Figure 3 shows no significant variation of R g (P,0) within the studied pressure range. The mean value of the actual radius of gyration of MbN 3 is R g (P,0) ¼ (1.52 ± 0.03) nm, in good agreement with the results of previous SANS studies of horse and sperm whale Mb at atmospheric pressure and finite concentrations [37,42]. Therefore, the reorganization of the secondary structure of Mb that has been observed by FTIR [23,24] does not affect the compactness of the protein. According to Eqn 5 the slope of the plot of c(P)[K 0 (P)] 2 / I(0,P,c)vs.c(P) allows the second virial coefficient, A 2 (P), to 0.0 0.2 0.4 0.6 0.8 q (nm –1 ) 0.15 0.20 0.25 0.30 0.35 0.40 0.45 I(q,P,c) (a.u.) Fig. 1. Scattering spectra I(q,P,c)ofMbN 3 at p 2 H 6.6, as a function of the wave-number transfer q. The measurements were performed at room temperature. The protein concentration, c, at atmospheric pressure is 11.7 mgÆcm )3 and the pressures, P,are:54(s), 154 (n), and 302 (h) MPa. Fits of Eqn 6 to the data are shown as full lines. 0 5 10 15 20 c (mg·cm -3 ) 0.35 0.40 0.45 0.50 0.55 [R g (P,c)] –2 (nm –2 ) 0 5 10 15 20 0.35 0.40 0.45 0.50 0.55 [R g (P,c)] –2 (nm –2 ) 0 5 10 15 20 0.35 0.40 0.45 0.50 0.55 [R g (P,c)] –2 (nm –2 ) C B A Fig. 2. Reciprocal of the square of the apparent radius of gyration, R g (P,c), as a function of MbN 3 concentration c(P). (A) P ¼ 54 MPa, (B) P ¼ 154 MPa, and (C) P ¼ 302 MPa. The solid lines are linear regressions. Ó FEBS 2002 Pressure effects on azidometmyoglobin (Eur. J. Biochem. 269) 4733 be determined. Figure 4 shows such plots for each studied pressure. The pressure dependence of A 2 (P) is given in Fig. 5. A 2 (P) decreases from 7.2 · 10 )4 cm 3 ÆmolÆg )2 at 54 MPa to 5.6 · 10 )4 cm 3 ÆmolÆg )2 at 302 MPa. The posi- tive values of A 2 (P) indicate that the interactions between two protein molecules are repulsive irrespective of the pressure. The second virial coefficient of a macromolecular solution can be estimated by means of the relation A 2 ðP Þ¼4p 3=2 wN A ½R g ðP ; 0Þ 3 M À2 p [43,44], where w is a constant depending on the shape and the conformation of the molecule. For hard spheres w ¼ 4pð5=3pÞ 3=2 =3 ¼ 1:619 [45]. If Mb molecules are regarded as hard spheres, the second virial coefficient would be close to 2.1 · 10 )4 cm 3 ÆmolÆg )2 . The much larger value of A 2 (P) inferred from the present SANS measurements at the lowest pressure is not due to the ellipsoid shape of Mb [37,42] but to the presence of electric charges on the protein surface and possibly, to a high surface hydration. Accordingly, the weakening of the repulsive interactions with increasing pressures can be attributed to either a decrease of the protein charge due to changes of the pKs of the side chains or a change of the protein hydration, or both these effects. As previously explained in Materials and methods, c(P) [K 0 (P)] 2 /I(0,P,c) has been calculated assuming that the partial specific volume of MbN 3 does not depend on the pressure and keeps the value v p (0.1) ¼ 0.741 cm 3 Æg )1 at atmospheric pressure. According to Eqn 5, {c(P)[K 0 (P)] 2 / I(0,P,c)} )1/2 extrapolated to c(P) ¼ 0 is proportional to the relative value of the actual protein contrast K(P)/K 0 (P). Figure 6 shows how this quantity vary with applied pressure. As no aggregation occurred during the experi- ments, any change in this ratio has to be ascribed to the variation of the average contrast of Mb with pressure and therefore to that of its specific volume v p (P). From the almost linear variation of v p (P) with pressure shown in Fig. 7, the values of both the specific volume, v p (0.1), at atmospheric pressure and the isothermal compressibility j T;p ¼À 1 v p ð0:1Þ ov p ðPÞ oP     T ð8Þ of hydrated Mb can be readily inferred. They are found to be v p (0.1) ¼ (0.741 ± 0.003) cm 3 Æg )1 and j T,p ¼ (1.6 ± 0.1) 10 )4 MPa )1 at about 20 °C. The value of v p (0.1) agrees well with that [40] used throughout the present analysis. DISCUSSION Previous studies on Mb under high hydrostatic pressures were performed by means of typical spectroscopic tech- niques that give information on the active site and on the secondary structure. All these investigations have shown that 0 5 10 15 20 c (m g ·cm –3 ) 2.0 2.4 2.8 3.2 3.6 c(P)[K 0 (P)] 2 /I(0,P,c) (a.u.) 0 5 10 15 20 2.0 2.4 2.8 3.2 3.6 c(P)[K 0 (P)] 2 /I(0,P,c) (a.u.) 0 5 10 15 20 2.0 2.4 2.8 3.2 3.6 c(P)[K 0 (P)] 2 /I(0,P,c) (a.u.) C B A Fig. 4. Plots of the quantity c(P)[K 0 (P)] 2 /I(0,P,c) as a function of the MbN 3 concentration c(P) at pressure P. I(0,P,c) is the forward scattered intensity and K 0 (P) the protein contrast defined by Eqn 4. K 0 (P) is calculated assuming that the partial specific volume of MbN 3 is independent of P and has an atmospheric pressure value v p (0.1) ¼ 0.741 cm 3 Æg )1 . According to Eqn 5, the slope of the solid regression lines is proportional to the second virial coefficient A 2 (P). (A) P ¼ 54 MPa, (B) P ¼ 154 MPa, and (C) P ¼ 302 MPa. 0 100 200 300 P (MPa) 1.2 1.3 1.4 1.5 1.6 1.7 1.8 R g (P,0) (nm) Fig. 3. Radius of gyration R g (P,0) of MbN 3 at p 2 H 6.6 at vanishing protein concentration as a function of pressure, P. 4734 C. Loupiac et al. (Eur. J. Biochem. 269) Ó FEBS 2002 moderate pressures near 300 MPa induce subtle structural rearrangements of the protein matrix whereas higher pressures, near 1 GPa, lead to unfolding. Pressure may also induce changes in the heme structure and in the spin state of the iron atom [17,18,20,21,26–29]. Other studies of proteins at high pressures have shown that they react as a whole, simultaneously adapting their structure, their spatial charge distribution and their interactions with the solvent [2,3]. The present SANS measurements on MbN 3 at pres- sures up to about 300 MPa indicate that the structural reorganization of the active site previously observed by optical absorption in the UV-visible range [17,18,20,21], Raman [26], and NMR [27–29] spectroscopies and the secondary structure modifications observed by FTIR through the amide I¢ band [23,24] are not related to a change of compactness of Mb as its radius of gyration remains constant. This does not means that MbN 3 remainsinthenativestateupto300MPa.Morelikely, the protein starts to denature at a lower pressure and becomes a slightly swollen molten globule. The value of the radius of gyration given by neutron scattering is indeed rather insensitive to the early stages of protein unfolding. This has been demonstrated for neocarzinos- tatine denatured by guanidinium chloride [46]. In the FTIR studies it was suggested that, in addition to the strengthening of the hydrogen bond network with increasing pressure, the bonding of a C¼O group with aN 2 H group and a water molecule may also occur. This means that the protein may become more hydrated with increasing pressure [24]. This increase of hydration might be due to the appearance of a molten globule state as the pressure dependence of the second virial coefficient suggests that the surface hydration decreases with pressure. The present SANS study allowed the specific volume of MbN 3 to be determined as a function of pressure. It decreases by about 5.4% between atmospheric pressure and 300 MPa. Within the uncertainties of the only three measurements carried out in this pressure range, the isothermal compressibility of hydrated MbN 3 is almost constant. Its value is j T,p ¼ (1.6 ± 0.1) 10 )4 MPa )1 at about 20 °C. Therefore, hydrated MbN 3 is about two to three times as incompressible as H 2 Oor 2 H 2 Oatthesame temperature. The value of the isothermal compressibility of hydrated MbN 3 compares well with that obtained by densimetry for staphylococcal nuclease at 25 °C: j T,p ¼ (1.1 ± 0.2) 10 )4 MPa )1 between atmospheric pres- sure and 60 MPa [47]. The above-mentioned values of the isothermal compress- ibility, j T,p , of proteins cannot be directly compared with those of the adiabatic compressibility, j S , inferred from ultrasound velocity measurements [48–53]. According to Eqn 8, j T,p is a characteristic property of the hydrated protein alone whereas j S is not as it is measured at constant 0 100 200 300 P (MPa) 0.63 0.65 0.67 0.69 {c(P)[K 0 (P)] 2 /I(0,P,c)} –1/2 (a.u.) Fig. 6. Plot of the quantity {c(P)[K 0 (P)] 2 /I(0,P,c)} )1/2 at vanishing protein concentration, c(P), as a function of pressure, P. I(0,P,c)isthe forward scattered intensity and K 0 (P) the protein contrast defined by Eqn 4. K 0 (P) is calculated assuming that the specific volume of MbN 3 is independent of P and has an atmospheric pressure value m p (0.1) ¼ 0.741 cm 3 Æg )1 .DataforMbN 3 at p 2 H6.6and20°C. 0 100 200 300 P (MPa) 0.700 0.710 0.720 0.730 0.740 0.750 v p (cm 3 g –1 ) Fig. 7. Partial specific volume v p (P), of MbN 3 as a function of pressure, P. The almost linear variation of m p (P)withP allows the isothermal compressibility of the hydrated protein to be computed: j T,p ¼ (1.6 ± 0.1) 10 )4 MPa )1 . 0 100 200 300 P (MPa) 5 6 7 8 A 2 (P) (10 –4 cm 3 mol g –2 ) Fig. 5. Second virial coefficient, A 2 (P), of MbN 3 at p 2 H6.6, as a function of pressure, P. Ó FEBS 2002 Pressure effects on azidometmyoglobin (Eur. J. Biochem. 269) 4735 entropy, S, of the solution. As a result j S is also sensitive to the thermodynamic properties of the solvent. Nevertheless, the value of j T,p can be inferred from that of j S if the densities, the thermal expansions and the specific heats at constant pressure of the solvent and the protein are known [53]. Once the value of j T,p is obtained, in this way or better still by means of densimetric or SANS measurements, it is possible to estimate the adiabatic compressibility, j S,p , characteristic of the hydrated protein. This compressibility at constant entropy of the hydrated protein is given by the standard thermodynamic expression j T;p ¼ j S;p þ Ta 2 p v p =C P;p ð9Þ where a p and C P,p are the thermal expansion and the specific heat at constant pressure of the hydrated protein, respec- tively. j T,p and j S,p are important quantities because their values provide an estimate of the magnitude of the different type of movements inside the hydrated protein. j S,p is the mean amplitude of the vibrational motions, or phonons, whereas (j T,p ) j S,p ) is that of the diffusive ones, associated with heat diffusion. This first SANS study of myoglobin at high hydro- static pressures shows that this approach not only gives global structural information about the protein molecule but also allows the protein–solvent interactions and the isothermal compressibility of the hydrated protein to be measured. Such information is important in order to understand the properties of proteins under pressure. 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