Piezotropic unfolding of lysozyme in pure D 2 O at the outer edge of excess hydration Helge Pfeiffer a,b, * , Karel Heremans b , Martine Wevers a a Department of Metallurgy and Materials Engineering, Katholieke Universiteit Leuven, Kasteelpark Arenberg 44, B-3001 Leuven, Belgium b Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, B-3001 Leuven, Belgium article info Article history: Received 18 November 2008 In final form 14 December 2008 Available online 25 December 2008 abstract This Letter reports a FTIR study on the pressure-induced unfolding of lysozyme in pure D 2 O close to the conditions of non excess hydration. An essential population of the proteins apparently refold into its native structure after pressure release. It could furthermore be shown that down to a hydration of h =2(h = m prot /m D 2 O ; m = mass), the unfolding pressure did not vary with hydration. Hydration depen- dent behaviour was found with respect to the change of the wavenumbers of the a-helical structure dur- ing unfolding. This result is discussed with respect to the reversibility of unfolding in pure solvents and with the effects of hydrogen exchange. Ó 2008 Elsevier B.V. All rights reserved. 1. Introduction This Letter addresses the issue of pressure-induced unfolding of hen egg lysozyme dissolved in pure D 2 O at relatively low hydration degree. This choice is based on at least three targets. Firstly, such as temperature, pressure is an important thermodynamic variable and pressure-tuning studies give a more extended access to the general understanding of the thermodynamic behaviour of pro- teins. Secondly, a very practical aspect emerges from high-pressure techniques for food preservation because pressure enables the deactivation of proteins being a prerequisite for the inactivation of harmful microorganisms. A molecular description of pressure- induced unfolding can contribute to the improved performance of those industrial processes, especially if temperature and pres- sure treatments can be combined. Last but not leased, almost all pressure-tuning unfolding studies published so far were performed at a quite high hydration degree, but excess hydration is not auto- matically guaranteed in native systems. If dehydration would e.g. stabilise the conformational stability of proteins, pressure treat- ment for food preservation could be more inefficient at low hydra- tion. Another aspect of investigating hydration phenomena regards the relationship between water activity and pressure on a more theoretical level. Pressure studies at different degree of hydration are therefore of special interest. A native protein is a polymeric macromolecule consisting of a defined sequence of amino acids (primary structure) displaying a unique folding pattern (secondary structure and higher order structures) which is characteristic for its native state. The initial folding into the native state can be visualised using a trajectory on a funnel shaped surface plot which represents the Gibbs-free- energy as a function of all possible conformational coordinates [1]. In its native state, the system exists at the bottom of that fun- nel, i.e. the minimum of the Gibbs-free-energy is adopted. If thermodynamic variables are changed, the shape of the fold- ing funnel also changes. A non-native global minimum can emerge, or the system can be trapped in local minima which also represent non-native states [2]. The native state of a protein is thus only guaranteed in a relatively narrow interval around the optimal val- ues of those thermodynamic variables for which structure and function of the protein were optimised during the respective trace of evolution. In the case of temperature and pressure, from all pos- sible pairs of pressure and temperature representing the native state a stability diagram can be constructed (see also Fig. 5 a) which is frequently described in the literature [3,4]. The measured data can be best fitted by a section of an elliptic curve; and the axes parameters give access to diverse thermodynamic quantities [3]. The elliptical stability diagram can be found not only for proteins, but also for further macromolecules such as different polymers, starch and even bacteria. The diagram is in its significance compa- rable to real phase diagrams, but without showing the same ther- modynamical severity. The reason is the inherent kinetic aspect of the transition between native and non-native states and the occur- rence of irreversible transitions. Moreover, not the real phase bor- ders are mostly given in the published diagrams but the coexistence lines when 50% of the respective phases are present. Deviations from the native folding pattern lead to diverse mal- functions that can result in a complete inactivation of the protein, even if the primary structure is maintained. In the case of physio- logical important proteins, severe illnesses can develop, such as the disease of Alzheimer. Proteins can thus exist in several non-native states, such as the unfolded, the inactivated or the aggregated state. The native state of a protein is self-explaining, the definition of the other states can be considered from different aspects. On the 0009-2614/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2008.12.063 * Corresponding author. Address: Department of Metallurgy and Materials Engineering, Katholieke Universiteit Leuven, Kasteelpark Arenberg 44, B-3001 Leuven, Belgium. Fax: +32 16 32 19 90. E-mail address: Helge.Pfeiffer@mtm.kuleuven.be (H. Pfeiffer). Chemical Physics Letters 469 (2009) 195–200 Contents lists available at ScienceDirect Chemical Physics Letters journal homepage: www.elsevier.com/locate/cplett