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

Taking advantage of the pressure-induced contrast variation, these experiments allow the partial specific volume of MbN3 to be determined as a function of pressure.. In this pressure rang

High-pressure effects on horse heart metmyoglobin studied

by small-angle neutron scattering

Camille Loupiac1, Marco Bonetti2, Serge Pin3and Patrick Calmettes1

1

Laboratoire Le´on Brillouin, UMR 12 CNRS,2Service de Physique de l’Etat Condense´, and3Service 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 (MbN3) 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 MbN3, 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 MbN3 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 MbN3is found to

be almost constant Its value is (1.6 ± 0.1) 10)4MPa)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 (2H2O) 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 (MbN3) 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]

M A T E R I A L S A N D M E T H O D S Protein sample preparation

High purity horse-heart Mb was purchased from Sigma The lyophilized protein was first dissolved in water (H2O) and dialysed three times against H2O during 24 h to remove all the salts The protein was then extensively dialysed (three dialyses of 24 h) against 2H2O to achieve a complete exchange of the labile hydrogen atoms For the SANS experiments a 0.1-MBistris p2H 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)

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 (NaN3) was added to the aquometmyoglobin

solution one day prior to the SANS experiments to ensure

that the protein was almost fully liganded with N3[36] This

was checked by absorbance measurements in the visible

region The p2H of the solution was measured after

ligandation and adjusted to 6.6 if necessary The mother

MbN3solution at about 60mgÆcm)3and the samples were

prepared and stored at 4C All samples were centrifuged at

20 000 g during 5 min at 15C 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 hmax¼ 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 k¼ 1.1 nm This

gives access to wavenumber transfers, q, ranging from 0.07

to 0.75 nm)1 q¼ (4p/k)sin(h/2), where h is the scattering

angle The SANS spectra were collected at room

temper-ature, near 20C 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; cÞ ¼ kBTcðPÞ½KðPÞ2oPðP; cÞ

oc

1 T;P ð1Þ

where kBis Bolztmann’s constant, T the temperature (K) and P the pressure (MPa) c¼ c(P) is the protein concen-tration (gÆcm)3) and P(P,c) the osmotic pressure (MPa), both at pressure P

KðPÞ ¼ bp

NA

Mp

 q0bðPÞvpðPÞ

ð2Þ

is the average specific contrast of the protein molecule with respect to the solvent bpis the coherent scattering length (cm) of a molecule and Mp its molar mass (gÆmol)1)

Mp@ 17.4 · 103gÆmol)1 for Mb in 2H2O [37] NA is Avogadro’s number vp(P) is the partial specific volume (cm3Æg)1) of the protein at pressure P q¢b(P) is the 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 pure2H2O Therefore its scattering length density is

q0bðPÞ ¼ b2 H 2 Oq2 H2OðPÞ NA

M2 H 2 O

ð3Þ

where b2 H 2 O is the coherent scattering length of a 2H2O molecule q2 H2O(P) and M2 H 2 O are the density (gÆcm)3) and the molar mass (gÆmol)1) of 2H2O, respectively At

20C, the pressure dependence of q2 H2O(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 H2O as a function of pressure [39] assuming that the molarities, q/M, of2H2O and H2O are identical at 20C 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 vp(0.1)¼ 0.741 cm3Æg)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

K0ðPÞ ¼ bp

NA

Mp

 q0bðPÞvpð0:1Þ

ð4Þ

where q0bðP Þ is given by Eqn 3

Using this expression and the virial expansion for the osmotic pressure, Eqn 1 can be rewritten as follows cðPÞ½K0ðPÞ2

Ið0;P;cÞ ¼

NA

Mp

K0ðPÞ KðPÞ

1þ 2A2ðPÞMpcðPÞ þ

ð5Þ where A2(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 A2(P) > 0for repulsive interactions between the solute molecules For relatively low solute concentrations, higher order terms in c(P) are negligible As both K0(P) and K(P) do not depend on c(P), a plot of c(P) [K0(P)]2/I(0,P,c) vs c(P) allows the value of A2(P) to

be determined The pressure dependence of the protein concentration was calculated by means of the expres-sion cðP Þ ¼ cð0:1Þq2 H 2 OðP Þ=q2 H 2 Oð0:1Þ, where c(0.1) and

q2 H2Oð0:1Þ 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 H2O

The SANS spectra from the protein were described by the

Guinier approximation [41]

Iðq; P; cÞ ffi Ið0; P; cÞ exp½q2R2gðP; cÞ=3 ð6Þ

where Rg(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 qRg(P,c) £ 1.3 [41] For a Mb

molecule, this corresponds to q £ 0.9 nm)1 All the SANS

spectra were collected within this range

The concentration dependence of the radius of gyration

can be accounted for by

½RgðP;cÞ2¼ ½RgðP; 0Þ2½1 þ 2B2ðPÞMpcðPÞþ 

ð7Þ where Rg(P,0) is the actual value of the radius of gyration of

the protein and B2(P) a constant similar to A2(P) in Eqn 5

but with a different value For each pressure, Rg(P,0) can be

inferred from the intercept of the plot of [Rg(P,c)])2as a

function of c(P)

R E S U L T S

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

the sample at 11.7 mgÆcm)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, Rg(P,c), of the MbN3molecule 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, Rg(P,0), at each pressure were inferred from

Rg(P,c) by extrapolation to c(P)¼ 0according to Eqn 7 Figure 3 shows no significant variation of Rg(P,0) within the studied pressure range The mean value of the actual radius of gyration of MbN3is Rg(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) [K0(P)]2/ I(0,P,c) vs c(P) allows the second virial coefficient, A2(P), to

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Fig 1 Scattering spectra I(q,P,c) of MbN 3 at p2H 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)3and the pressures, P, are: 54 (s), 154 (n), and

302 (h) MPa Fits of Eqn 6 to the data are shown as full lines.

c (mg·cm-3)

0.35 0.40 0.45 0.50 0.55

0.35 0.40 0.45 0.50 0.55

0.35 0.40 0.45 0.50 0.55

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.

be determined Figure 4 shows such plots for each studied

pressure The pressure dependence of A2(P) is given in

Fig 5 A2(P) decreases from 7.2· 10)4cm3ÆmolÆg)2 at

54 MPa to 5.6· 10)4cm3ÆmolÆg)2at 302 MPa The

posi-tive values of A2(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

A2ðP Þ ¼ 4p3=2wNA½RgðP ; 0Þ3M2

p [43,44], where w is a constant depending on the shape and the conformation of

the molecule For hard spheres w¼ 4pð5=3pÞ3=2= ¼

1:619 [45] If Mb molecules are regarded as hard spheres,

the second virial coefficient would be close to 2.1·

10)4cm3ÆmolÆg)2 The much larger value of A2(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)

[K0(P)]2/I(0,P,c) has been calculated assuming that the

partial specific volume of MbN3does not depend on the

pressure and keeps the value vp(0.1)¼ 0.741 cm3Æg)1 at

atmospheric pressure According to Eqn 5, {c(P)[K0(P)]2/

I(0,P,c)})1/2extrapolated to c(P)¼ 0is proportional to the

relative value of the actual protein contrast K(P)/K0(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 vp(P) From the

almost linear variation of vp(P) with pressure shown in

Fig 7, the values of both the specific volume, vp(0.1), at

atmospheric pressure and the isothermal compressibility

jT;p¼  1

vpð0:1Þ

ovpðPÞ oP

T

ð8Þ

of hydrated Mb can be readily inferred They are found

to be vp(0.1)¼ (0.741 ± 0.003) cm3Æg)1and jT,p¼ (1.6 ± 0.1) 10)4MPa)1at about 20C The value of vp(0.1) agrees well with that [40] used throughout the present analysis

D I S C U S S I O N 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

c (mg·cm–3) 2.0

2.4 2.8 3.2 3.6

2 /I(0,P,c) (a.u.)

2.0 2.4 2.8 3.2 3.6

2 /I(0,P,c) (a.u.)

2.0 2.4 2.8 3.2 3.6

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.

P (MPa)

1.2

1.3

1.4

1.5

1.6

1.7

1.8

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.

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 MbN3 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 MbN3 remains in the native state up to 30 0 MPa More likely, 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

a N2H 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 MbN3 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 MbN3 is almost constant Its value is jT,p¼ (1.6 ± 0.1) 10)4MPa)1 at about 20C Therefore, hydrated MbN3 is about two to three times as incompressible as H2O or2H2O at the same temperature The value of the isothermal compressibility of hydrated MbN3 compares well with that obtained by densimetry for staphylococcal nuclease at 25C:

jT,p¼ (1.1 ± 0.2) 10)4MPa)1between atmospheric pres-sure and 60MPa [47]

The above-mentioned values of the isothermal compress-ibility, jT,p, of proteins cannot be directly compared with those of the adiabatic compressibility, jS, inferred from ultrasound velocity measurements [48–53] According to Eqn 8, jT,p is a characteristic property of the hydrated protein alone whereas j is not as it is measured at constant

P (MPa) 0.63

0.65

0.67

0.69

2 /I(0,P,c)}

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) is the

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 (0.1) ¼ 0.741 cm 3 Æg)1 Data for MbN at p 2 H 6.6 and 20 C.

P (MPa)

0.700 0.710 0.720 0.730 0.740 0.750

vp

3 g

– )

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) with P allows the isothermal compressibility of the hydrated protein to be computed: j T,p ¼ (1.6 ± 0.1) 10)4MPa)1.

P (MPa)

5

6

7

8

3 mol g

Fig 5 Second virial coefficient, A 2 (P), of MbN 3 at p2H 6.6, as a

function of pressure, P.

entropy, S, of the solution As a result jSis also sensitive to

the thermodynamic properties of the solvent Nevertheless,

the value of jT,p can be inferred from that of jS 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 jT,pis obtained, in this way or better

still by means of densimetric or SANS measurements, it is

possible to estimate the adiabatic compressibility, jS,p,

characteristic of the hydrated protein This compressibility

at constant entropy of the hydrated protein is given by the

standard thermodynamic expression

jT;p¼ jS;pþ Ta2

pvp=CP;p ð9Þ where apand CP,pare the thermal expansion and the specific

heat at constant pressure of the hydrated protein,

respec-tively

jT,pand jS,pare important quantities because their values

provide an estimate of the magnitude of the different type of

movements inside the hydrated protein jS,p is the mean

amplitude of the vibrational motions, or phonons, whereas

(jT,p) jS,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 In

the future it would be beneficial to perform SANS

measurements at higher pressures in order to determine

the properties and the conformations of the various

denatured states

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the direct neutron beam Consequently, no significant

increase of the scattered intensity is observed for the

smallest q-values This demonstrates... 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... Calmettes, P & Desmadril, M (2001) Characterization of the denatured states distribution of neocarzi-nostatin by small-angle neutron scattering and differential scan-ning calorimetry Biochemistry