High‐pressure effects on horse heart metmyoglobin studied by small‐angle neutron scattering

Abstract

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 pressureinduced 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 application of hydrostatic pressure to a protein solution also provides a manner to alter these physical properties [1][2][3][4]. Therefore, it is important to determine whether the modifications observed at the level of the active site of myoglobin [17,18,20,21,[26][27][28][29] and the reorganization of the secondary structure with an alteration of the electrostatic and hydrogen-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 measurements were carried out in heavy water ( 2 H 2 O) at varying pressures up to 300 MPa as a function of protein concentration 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].

Refolding of proteins from inclusion bodies is favored by a diminished hydrophobic effect at elevated pressures

“…In contrast, at 1,000 bar pressure, there is a monotonic increase in B 22 values as a function of GdnHCl concentration. In agreement, using small-angle neutron scattering, Loupiac et al (2002) also showed positive B 22 values at pressures up to 3,000 bar for horse azidometmyoglobin. Surprisingly, the effect of pressure on protein--protein interactions is independent of the conformational stability of these proteins, in contrast to results shown at atmospheric pressure (Chi et al, 2003a).…”

Comparison of heat- and pressure-induced gelation of β-lactoglobulin aqueous solutions studied by small-angle neutron and dynamic light scattering

“…This conjecture is consistent with the model discussed by LopezFandino [12]. It is reported that tryptophan residues in bLG in an aqueous solutions become part of a more hydrophilic by pressurizing [52] and the positive virial coefficient of the bLG decreases [53]. These results mean that the affinity of the protein to water increases by pressurizing.…”

A high pressure cell using metallic windows to investigate the structure of molecular solutions up to 600 MPa by small-angle neutron scattering

“…5(b)], the SANS intensity was virtually the same all along the pressure increase, in agreement with the data obtained on this protein in similar conditions, but with pressures limited to 300 MPa, using a HP cell with sapphire windows. 5,6 Here, we show that the absence of variation was confirmed up to 500 MPa. The behavior of the protein solution under HP was significantly different compared to aquometMb [ Fig.…”

“…Many studies have been published using SANS and HP on different proteins. For instance, measurements performed on various concentrations of azidometmyoglobin (myoglobin being a small and globular protein, whose function is to stock dioxygen in muscles) showed that the compactness of the protein is not altered by HP up to 300 MPa (or 3 kbar), 6 but some changes have however been observed on the specific volume of this protein as a function of pressure. 7 Ortore et al reported that pressure induces dissociation of β-lactoglobulin in both D 2 O and a 50% mixture of water and ethylene-glycol, even if the protein shows a higher stability in 50% ethyleneglycol.…”