Influence of pressure on structure and dynamics of bovine pancreatic trypsin inhibitor (BPTI): Small angle and quasi-elastic neutron scattering studies

https://doi.org/10.1016/j.bbapap.2006.01.010Get rights and content

Abstract

We have studied the influence of pressure on structure and dynamics of a small protein belonging to the enzymatic catalysis: the bovine pancreatic trypsin inhibitor (BPTI). Using a copper-beryllium high-pressure cell, we have performed small angle neutron scattering (SANS) experiment on NEAT spectrometer at HMI (Berlin, Germany). In the SANS configuration, the evolution of the radius of gyration and of the shape of the protein under pressures up to 6000 bar has been studied. When increasing pressure from atmospheric pressure up to 6000 bar, the pressure effects on the global structure of BPTI result on a reduction of the radius of gyration from 13.4 Å down to 12.0 Å. Between 5000 and 6000 bar, some transition already detected by FTIR [N. Takeda, K. Nakano, M. Kato, Y. Taniguchi, Biospectroscopy, 4, 1998, pp. 209–216] is observed. The pressure effect is not reversible because the initial value of the radius of gyration is not recovered after pressure release. By extending the range of wave-vectors to high q, we have observed a change of the form factor (shape) of the BPTI under pressure. At atmospheric pressure BPTI exhibits an ellipsoidal form factor that is characteristic of the native state. When the pressure is increased from atmospheric pressure up to 6000 bar, the protein keeps its ellipsoidal shape. The parameters of the ellipsoid vary and the transition detected between 5000 and 6000 bar in the form factor of BPTI is in agreement with the FTIR results. After pressure release, the form factor of BPTI is characteristic of an ellipsoid of revolution with a semi-axis a, slightly elongated with respect to that of the native one, indicating that the pressure-induced structural changes on the protein are not reversible. The global motions and the internal dynamics of BPTI protein have been investigated in the same pressure range by quasi-elastic neutron scattering experiments on IN5 time-of-flight spectrometer at ILL (Grenoble, France). The diffusion coefficients D and the internal relaxation times <τ2> of BPTI deduced from the analysis of the intermediate scattering functions show a slowing down of protein dynamics when increasing pressure.

Introduction

Most of recent studies about pressure effects on biological systems have been mentioned in general publications by Silva and Weber [1] in 1993, by Prehoda et al. [2] in 1997, by Heremans and Smeller [3] in 1998, by Mentré and Hui Bon Hoa [4] in 2000, and by Heremans [5] in 2004 for the more recent publication. A collection of reviews edited by Balny, Masson and Heremans [6] in Biochemica et Biophysica Acta in 2002 brings together a large panel of studies realised with several techniques and on several proteins.

The study of the influence of temperature and pressure on proteins has a practical interest because these two parameters are employed for sterilization process and bioconservation in food and pharmaceutical industries. From a biological point of view, the influence of pressure on proteins is interesting because in physiological media these macromolecules can be submitted at pressures from a few millibar in blood circulation up to nearly 1000 bar in Deep Ocean. Organisms called barophiles can support pressure values up to 5000 bar like Bacillus licheniformis [7].

Concerning the protein folding investigation, pressure can induce unfolding pathways different from thermal unfolding. Instead of temperature that induces changes in the reaction volume and in the thermal energy, pressure affects only the reaction volume. This makes the thermodynamic of unfolding more easy to be followed. Moreover, pressure unfolding is accompanied by a reorganisation of hydration water at the surface of proteins.

Structural studies have been done by small angle neutron scattering (SANS) technique that gives information about the global structure of molecules as proteins. In the Guinier regime, one gets the radius of gyration and in the Porod regime, the specific surface of molecule. In order to get the influence of pressure on radius of gyration and on intermolecular interactions of horse heart metmyoglobin, SANS experiments were performed at pressures up to 3000 bar by Loupiac et al. [8]. The evolution of the specific molar volume of metmyoglobin as a function of applied pressure has been determined by this technique. It has been shown that the specific molar volume of this protein decreases by a factor of 5.4% in this range of pressure. Moreover, the authors have shown that the radius of gyration of metmyoglobin remains constant and that, between atmospheric pressure and 3000 bar, the intermolecular interactions are always repulsive [8]. In order to study the influence of pressures up to 3000 bar on the structure of staphylocoque nuclease, Paliwal et al. [9] have used small angle neutron scattering in complement of molecular dynamics simulation. They have observed the folding–unfolding transition of the protein. When the pressure is applied, a two-step increase of the radius of gyration and a modification of the shape of the protein are obtained. At the highest pressure, the protein is less compact and more elongated than in the native state.

In protein, several motions occur on a wide time scale, from femtosecond for electronic transitions, to picosecond and nanosecond for vibrational and diffusive motions, and millisecond for conformational changes, and second to minute for kinetics. Incoherent quasi-elastic neutron scattering allows us to explore individual motions from picosecond to nanosecond time scale [10]. Using this technique, the markers of protein motion are the hydrogen atoms (due to the anomalously large incoherent neutron scattering cross-section of the 1H nucleus) that are uniformly distributed throughout the protein. The quantity measured is the incoherent dynamic structure factor Sinc(q, ω) which is the space-time Fourier transform of the self-correlation of the position of an atom at time 0 with the position of the same atom at time t.

The influence of pressure between atmospheric pressure and 900 bar on the internal dynamics of trypsin in solution [11] has been recently studied. The authors have shown a diminution of the volume inside which the internal motions of non-labile protons occur and a narrowing of dynamic structure factor Sinc(q, ω) showing a slowing down of internal dynamics of trypsin. In 2003, Doster and Gebhardt [12] have observed the influence of pressure up to 7000 bar on internal dynamics of myoglobin by quasi-elastic neutron scattering. The authors have observed a slowing down of the dynamics with a transition at 3000 bar. Beyond this pressure, myoglobin does not keep its native structure [8].

Our interest focused on BPTI protein that is a small protein belonging to the enzymatic catalysis. Its function is to inhibit protease-like trypsin by inserting its lysine 15 inside the catalytic site. It is a small protein composed by 58 amino acid residues and with a molecular weight of about 6500 Da. Its crystallographic structure is well known at 1.7 Å resolution [13]. A particularity of this protein is the presence of three disulphide bridges and three salt bridges that induce a very high stability throughout temperatures up to 95 °C and pressures up to 14 kbar. This model system was studied at high temperature by differential scanning calorimetry [14] and Raman spectroscopy [15]. BPTI begins to be unfolded above 95 °C. The influence of pressure on BPTI in solution has been studied by FTIR [16], [17] showing that some secondary structures still exist at 14 kbar.

In this paper, we present results from small angle neutron scattering performed on NEAT spectrometer at HMI (Berlin) and from quasi-elastic neutron scattering on IN5 spectrometer at ILL (Grenoble), on a 85 mg/ml concentrated solution of BPTI, as a function of applied pressure between 1 bar and 6000 bar and after pressure release.

Section snippets

Sample preparation

BPTI was purchased from SIGMA-ALDRICH (A-4529) as a lyophilised powder and used without any further purification. Deuterated acetic acid and D2O were purchased from EURISOTOP. BPTI lyophilised powder is dissolved overnight at room temperature in a deuterated acetic acid buffer at 50 mM concentration in D2O with 100 mM ammonium sulphate salts. The solution of BPTI is filtered through a 0.22 μm pore diameter filter, then dialysed against the same buffer during 24 or 48 h in order to complete H/D

Small angle neutron scattering experiments

A concentrated solution of BPTI (85 mg/ml) was studied by small angle neutron scattering, as a function of pressure, on NEAT spectrometer at HMI (Berlin).

In order to estimate the structure factor S(q) at high concentration (85 mg/ml), we have calculated the ratio between the intensity I(q) of BPTI at high concentration divided by that of BPTI at low concentration. If we assume that the form factor of BPTI is independent of the concentration, this ratio is equal to S(q) times the ratio of the

Small angle neutron scattering experiment

At atmospheric pressure, the form factor of BPTI as deduced from the SANS spectrum is in agreement with the crystallographic structure (6PTI, [13]) and can be accounted for by an ellipsoidal one with a volume of 8400 Å3. At 3000 bar, BPTI gets a more elongated ellipsoidal shape than in the native state; the parameters of the ellipsoid of revolution have slightly changed, in particular the value of semi-axis a (Table 4). This finding is in agreement with the N.M.R. results at 2000 bar by

Conclusion

In the frame of a biophysical approach of protein denaturation by pressure, we have used a high-pressure cell made with a copper-beryllium alloy in order to investigate the influence of this thermodynamic parameter on the structure and the dynamics of a model protein, such as BPTI. Structural investigation by small angle neutron scattering on a BPTI solution at 85 mg/ml has shown the following evolution. Between atmospheric pressure and 6000 bar, the protein keeps its ellipsoidal shape with

Acknowledgements

We would like to thank very much all our local contact and technicians who help us for performing neutron scattering experiments: Jörg Pieper and Alexandra Buschteiner on NEAT (V3) time-of-flight and small angle neutron scattering spectrometer at HMI (BENSC, Berlin, Germany). We would like to thank Burkhard Annighöfer (LLB, Saclay, France) who realised the Cu–Be high pressure cell, Marie Plazanet and Steve Jenkins on IN5 time-of-flight neutron scattering spectrometer at ILL (Grenoble, France)

References (30)

  • C. Balny et al.

    High pressure effects on biological macromolecules: from structural changes to alteration of cellular processes

    Biochim. Biophys. Acta

    (2002)
  • C. Balny

    Bull. Soc. Fr. Biophys.

    (2003)
  • C. Loupiac et al.

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

    Eur. J. Biochem.

    (2002)
  • J.A. McCammon et al.
  • M. Di Bari et al.

    Dynamics of trypsin under pressure

    Physica B

    (2000)
  • Cited by (0)

    View full text