The impact of high hydrostatic pressure on structure and dynamics of β-lactoglobulin Daniela Russo a,b, ⁎, Maria Grazia Ortore c,d, ⁎, Francesco Spinozzi c,d , Paolo Mariani c,d , Camille Loupiac e , Burkhard Annighofer f , Alessandro Paciaroni g, ⁎ a CNR-IOM, c/o Institut Laue-Langevin, Grenoble, France b Institut Lumière Matière, Université de Lyon 1, France c Department of Life and Environmental Sciences, Marche Polytechnic University, Ancona, Italy d CNISM, Ancona, Italy e PAPC, UMR PAM, AgroSup Dijon-Université de Bourgogne, Dijon, France f Laboratoire Léon Brillouin, Saclay CEA, Paris, France g Department of Physics, University of Perugia, Perugia, Italy abstract article info Article history: Received 14 December 2012 Received in revised form 6 June 2013 Accepted 29 June 2013 Available online 10 July 2013 Keywords: Hydrostatic pressure Protein folding Protein dynamics Neutron scattering Small angle X-ray and neutron scattering Methods: Combining small-angle X-ray and neutron scattering measurements with inelastic neutron scattering ex- periments, we investigated the impact of high hydrostatic pressure on the structure and dynamics of β-lactoglobulin (βLG) in aqueous solution. Background: βLG is a relatively small protein, which is predominantly dimeric in physiological conditions, but dis- sociates to monomer below about pH 3. Results: High-pressure structural results show that the dimer–monomer equilibrium, as well as the protein–protein interactions, are only slightly perturbed by pressure, and βLG unfolding is observed above a threshold value of 3000 bar. In the same range of pressure, dynamical results put in evidence a slowing down of the protein dynamics in the picosecond timescale and a loss of rigidity of the βLG structure. This dynamical behavior can be related to the onset of unfolding processes, probably promoted from water penetration in the hydrophobic cavity. General significance: Results suggest that density and compressibility of water molecules in contact with the pro- tein are key parameters to regulate the protein flexibility. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Pressure effects on conformational, dynamic and solvation properties of proteins in solution are nowadays a noticeable matter of debate [1–5]. While in the last decades the application of high hydrostatic pressures to proteins in solution has been an exotic method to investigate protein thermodynamic stability, now the protein response to not-denaturing pressures draws new interests. In fact, moderate values of hydrostatic pressure can provide slight modifications in protein–protein interactions [2,4,6], in solvation processes [4,5], in the population of conformational substates [7] as well as in the aggregational states of a protein [8,9]. By increasing pressure, the protein states or substates that lead to a lower partial volume of the solution are more populated. Due to the water electrostriction effect, usually these states are the ones that expose a higher surface toward the solvent. Hence, among the external variables that can be finely tuned, such as temperature, concentration or pH, pressure is a parameter that can be easily changed in order to gain insight over structural and dynamic processes of a protein driven only by volumetric effects. In particular, protein–protein interactions in con- centrated lysozyme solutions (≃ 10 wt.%) have been demonstrated to be affected by pressure even in the biological relevant pressure range (i.e. at pressures lower than 1100 bar), before the activation of unfolding processes [2,4,5]. It is also known that pressure affects the biological activity of a protein, which in native conditions is directly connected not only to the structure but also to the dynamic motions of the molecule [10–16]. Hence, from a biophysical point of view another important issue to be investigated is the relationship between pressure and protein dynamics at different time scales. In this framework, high-pressure treatments of milk proteins are of considerable interest [17]. Among milk proteins, β-lactoglobulin (βLG) has become a good model to study mechanical pressure effects with different experimental and theoretical approaches. Indeed, this small protein, belonging to the family of proteins that transport small fatty molecules (the lipocalin family), can be easily obtained from mammalian milk and purified in moderately high amount, allowing to be investigated by very different techniques in a large range of concentrations. Moreover, it is known that βLG solutions show a monomer/dimer equilibrium, which can be easily modified by small variations of concentration, pH and/or ionic strength. It has been observed that the βLG unfolding under 3500 bar of pressure is Biochimica et Biophysica Acta 1830 (2013) 4974–4980 ⁎ Corresponding authors. Tel.: +33 476207683. E-mail addresses: russo@ill.fr (D. Russo), m.g.ortore@univpm.it (M.G. Ortore), alessandro.paciaroni@fisica.unipg.it (A. Paciaroni). 0304-4165/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbagen.2013.06.040 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen