Low frequency enzyme dynamics as a function of temperature and hydration: A neutron scattering study

doi:10.1016/j.chemphys.2005.05.019

Chemical Physics

Volume 317, Issues 2–3, 31 October 2005, Pages 267-273

https://doi.org/10.1016/j.chemphys.2005.05.019 Get rights and content

Abstract

The effect of hydration and temperature on the low-frequency dynamics of the enzyme Pig liver esterase has been investigated with incoherent neutron scattering experiments. The results suggest that at low temperature, increasing hydration results in lower flexibility of the protein. At higher temperatures, systems containing sufficient number of water molecules interacting with the protein exhibit increased flexibility. The environmental force constants indicate that the environment of the protein is more rigid below than it is above the dynamical transition temperature.

Introduction

The elucidation of protein:water interactions is required for a complete understanding of protein stability, folding and function. It is generally accepted that protein hydration is essential for enzyme catalysis to occur [1], [2], [3], [4], [5], and that dry enzymes are inactive [6], [7].

There have been numerous experimental and theoretical studies of protein hydration water [8], [9], [10], [11], [12], [13], [14], [15], [16], [17]. Detailed information on the ordering and dynamical properties of individual, highly perturbed, strongly-bound water molecules has been furnished by high-resolution X-ray crystallography and NMR spectroscopy [18], [19], [20], [21], [22]. Averaged statistical properties of the solvent have been derived using molecular dynamics (MD) simulations [23] and solution scattering [24]. Two classes of hydration water may be defined. The first consists of individual, internal water molecules that may be reactants in a catalysed reaction and/or may be integral parts of a protein structure, making stereospecific interactions with the protein [3], [25], [26], [27]. The second class is the ubiquitous surface hydration shell that is believed to have a significant influence on protein structure, dynamics and function. It is the influence of this second category of water molecules on protein dynamics which is the subject of the present work. It also has been reported that the influence of hydration depends on the solvent used [28]; for example the dynamical transition in myoglobin is suppressed when trehalose is used as a solvent [29].

Incoherent neutron scattering (INS) is a particularly useful tool for determining hydration-dependent dynamics of biomolecules. INS probes motions on length scales of the order of Å and timescales ranging from femtoseconds to nanoseconds [30]. In the present work, we probe the hydration and temperature dependence of low-frequency protein dynamics. Neutron scattering experiments are performed on four different samples of pig liver esterase at hydrations of 0%, 3%, 12% and 50% and at temperatures ranging from 120 to 295 K. Since the incoherent scattering cross-section of the hydrogen atom is an order of magnitude larger than that of the other atoms present in the system, the measured spectrum is dominated by the incoherent single-particle hydrogen scattering.

The rest of this article is organised as follows. Section 2 provides the methods used for sample preparation and the procedure for data reduction followed by the results in Section 3. Section 4 offers discussions of the results and some concluding remarks.

Section snippets

Sample preparation

Pig liver esterase (150 units/mg, EC 3.1.1.1) was obtained from Sigma, and further partially purified using Fast Flow Q Sepharose. The enzyme was hydrogen/deuterium exchanged by twice dissolving the protein in 99.9% D₂O at 10 mg/ml, for 20 h at 4 °C, and then lyophilised.

A completely anhydrous enzyme powder was prepared by extensive drying in the aluminium sample holder. The lyophilised powder was initially packed into the sample holder at 4 °C to prevent back exchange. The sample was then placed

Results

Fig. 1, Fig. 2 show the normalised structure factor S(q_mean, E)_norm as a function of energy transfer E at temperatures of 120 and 295 K.

The feature often seen in the low-frequency regime of inelastic neutron scattering and Raman spectra is often termed as the “Boson peak”. This peak has been observed in both experimental investigations [11], [37], [38] and MD simulations of a range of disordered systems [16], [37], [39]. For the 120 K results (Fig. 1), the Boson peak intensity decreases and the

Discussions and conclusions

Quasielastic scattering, which appears as a broadening under the elastic peak, is due to diffusive motion present in the system. As the vanadium sample is a pure elastic scatterer, any broadening of the sample spectrum with respect to that of the vanadium is a sign of quasielastic scattering in the sample. The present results indicate that quasielastic scattering is present both in the dry and wet samples at a temperature as low as 120 K.

At 120 K, the Boson peak intensity decrease observed with

Acknowledgements

The authors thank Institut Laue-Langevin, Grenoble for support and Jacques Ollivier for his assistance as local contact during the neutron scattering experiments. V.K. and J.C.S. acknowledge DFG for support. V.K. acknowledges the financial support through Olympia Morata fellowship. M.T., R.V.D. and R.M.D. acknowledge support by the Marsden Fund of the Royal Society of New Zealand.

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¹: Present address: CRGIN13, Institut Laue-Langevin, 6 rue Jules, Horowitz BP 156, 38042 Grenoble Cedex 9, France.

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Low frequency enzyme dynamics as a function of temperature and hydration: A neutron scattering study

Abstract

Introduction

Section snippets

Sample preparation

Results

Discussions and conclusions

Acknowledgements

J. Mol. Biol

Prog. Biophys. Mol. Biol

Adv. Protein Chem.

Chem. Phys.

Biophys. J

Biophys. J.

Biophys. J.

Biophys. J.

Biophys. J.

Biophys. J.

Biophys. J.

Q. Rev. Biophys.

Biophys. J.

Chem. Phys.

J. Chem. Phys.

Chem. Phys. Lett.

Chem. Phys.

Adv. Food Res.

Biotechnol. Bioeng.

Appl. Biochem. Biotechnol.

Faraday Discuss.