Study of phase transition in tetragonal lysozyme crystals by AC-nanocalorimetry

Abstract

Differential ac-nanocalorimetric studies of a structural phase transition in the vicinity of 306 K in tetragonal lysozyme crystals have been carried out. It was found that the anomalies in the temperature dependences of heat capacity of the crystals look like λ-type anomalies. On the other hand, a pronounced temperature hysteresis has been observed at temperature cycling. The variation of experimental conditions such as relative humidity, frequency of the AC heat flow applied to the crystal of the order of 10 Hz, and heating rate seem not to affect the temperature of the anomaly in C_p(T).

Introduction

All biochemical processes in living organisms involve the participation of biopolymers, such as proteins, DNA, RNA, and others. Structural rearrangements and changes in dynamics of biopolymers are caused by interactions with ligands [1] or drugs [2], variations in temperature [3], [4] or pressure [5] and other factors. All this can lead to changes in biopolymer activities and functioning. For this reason phase transformations in biopolymers and proteins, in particular, are the objects of intense investigations. Studies of protein crystals give information not only on their structures but also on physical properties and on their phase transitions. It is known that phase transitions in protein crystals can occur at dehydration [6], [7] which is accompanied by changes in the protein crystal structure and molecular conformational rearrangements. Recently it has been found that the structural phase transition in tetragonal lysozyme crystals at about 305 K is induced by a temperature increase [8].

For the first time a structural phase transition in protein crystals at heating was discussed by Jolles et al. [9]. It was shown that when tetragonal lysozyme crystals with space group P4₃2₁2 were heated in a mother solution, their symmetry became orthorhombic P2₁2₁2₁ in the vicinity of 298 K. Later it was found that optical properties of tetragonal lysozyme crystals changed at increasing temperature [10]. Namely, Kobayashi et al. showed that the temperature dependence of the birefringence index of lysozyme crystals exhibited a step-like anomaly at 306.5 K. Investigations of lattice dynamics of tetragonal lysozyme crystals by Brillouin light scattering and ac-nanocalorimetry revealed anomalies in the vicinity of 306 K in the temperature behaviors of both the heat capacity and hypersonic acoustic phonons [8]. All these facts indicate that a structural phase transition occurs in tetragonal lysozyme crystals in the vicinity of 305 K.

One of the techniques that offers great opportunities for the research into the phase transition nature in condensed matter is calorimetry [11], [12], [13], [14], [15], [16]. For example, differential scanning calorimetry has already become a universal tool for studies of phase transitions and conformational changes in biopolymers [4], [13]. However, investigation of protein crystals by adiabatic, relaxational, etc., calorimetry encounters difficulties due to small crystal sizes (0.2–0.5 mm) and the effect of environmental conditions on the crystal state. In order to keep a protein crystal in the stable state, a definite humidity in the environment should be maintained, otherwise crystal drying can lead to its degradation. These problems can be overcome by using AC-nanocalorimetry [14] that allows highly sensitive measurements of the complex heat capacity in a broad frequency range. In order to maintain the optimal crystal state, a special AC-nanocalorimeter with controlled humidity in the sample chamber was constructed. AC-nanocalorimetric studies of the lattice dynamics of lysozyme crystals revealed heat capacity anomalies attributed to a structural phase transition [8]. The goal of the present work was to clarify the nature of the observed phase transition in lysozyme crystals. Thermal analysis of the crystals was done by AC-nanocalorimetry at different relative humidities in the experimental chamber, different frequencies of the applied heat flow, and sample heating rates at heating and cooling.