Effect of conformational states on protein dynamical transition

Abstract

In order to examine the properties specific to the folded protein, the effect of the conformational states on protein dynamical transition was studied by incoherent elastic neutron scattering for both wild type and a deletion mutant of staphylococcal nuclease. The deletion mutant of SNase which lacks C-terminal 13 residues takes a compact denatured structure, and can be regarded as a model of intrinsic unstructured protein. Incoherent elastic neutron scattering experiments were carried out at various temperature between 10 K and 300 K on IN10 and IN13 installed at ILL. Temperature dependence of mean-square displacements was obtained by the q-dependence of elastic scattering intensity. The measurements were performed on dried and hydrated powder samples. No significant differences were observed between wild type and the mutant for the hydrated samples, while significant differences were observed for the dried samples. A dynamical transition at ∼ 140 K observed for both dried and hydrated samples. The slopes of the temperature dependence of MSD before transition and after transition are different between wild type and the mutant, indicating the folding induces hardening. The hydration water activates a further transition at ∼ 240 K. The behavior of the temperature dependence of MSD is indistinguishable for wild type and the mutant, indicating that hydration water dynamics dominate the dynamical properties.

Introduction

It is widely accepted that the unique tertiary structure of a protein is required for its specific function. The structure–function relationship is thus a central subject in protein science even in the post-genomic era. Many studies have demonstrated that the internal protein motion is required for the function [1], [2], [3], [4], [5]. Therefore, there arises the question whether a protein acquires the specific dynamical properties upon folding. The dynamical differences between a native and non-native proteins have been characterized [6], [7], [8], [9], [10], [11], [12]. These studies suggest that the high-frequency, bond-vibrational and angle-bending motions are almost identical regardless of their conformations [13], [14], whereas the lower-frequency collective dynamics are different. An additional factor to be considered is the solvent. Although the potential surface of a protein in vacuo is intrinsically anharmonic [15], it is also accepted that solvent induces the dynamical transition from harmonic to anharmonic dynamical behavior in globular proteins i.e., protein dynamics are strongly coupled to their solvent environment [16], [17], [18], [19], [20], [21], [22], [23].

Incoherent neutron scattering (INS) is useful and effective for the studies of the internal dynamics of proteins in the picosecond–nanosecond time scale [24]. INS probes the self time-correlations of the hydrogen atom positions sensitively due to its large incoherent cross section. Because hydrogen atoms are evenly distributed throughout a protein, this technique gives a global view of the protein motions. The dependence of elastic incoherent scattering intensity on the scattering vector (q) provides the average mean-square displacements (MSD) in proteins [17], [25]. Derivation of the MSD requires the assumption that the elastic scattering is approximated by a Gaussian function [23], [26]. It is usually valid the Gaussian approximation in low q region, but in wide q range, the deviation from a Gaussian becomes remarkable [27], [28]. The non-Gaussian behavior can occur from the individual atoms exhibiting the non-Gaussian dynamics (such as jump diffusion) and/or by the existence of more than one MSD value in the proteins, called as the dynamical heterogeneity [15], [29]. We reported that the dynamical heterogeneity dominantly contributes to the non-Gaussian behavior [29], [30], [31]. The dynamical heterogeneity leads to the non-Gaussian scattering even when the individual atom scattering is a Gaussian [15], [32]. We have also shown that a bimodal distribution is adequate for the quantitative analysis of the experimentally observed scattering function in terms of dynamical heterogeneity, by which the protein internal motions can be separated into the inner core and surface part in the protein structure [29].

The time scale of the motions observed by INS is determined by the instrumental energy resolution [33], [34]. Protein motions can be observed when the time scale of the motions is within the instrumental frequency windows. Daniel et al. showed experimentally that the observed dynamics of the enzyme, glutamate dehydrogenase (GDH), possesses a marked dependence on the time scale [35]. The molecular dynamics simulations demonstrated that the observed dynamical transition is varied by the instrumental energy resolution [33], [35].

We focus here on the comparison of dynamics between a folded and partially unfolded protein, Staphylococcal nuclease (SNase). The partial unfolding was achieved by the truncation of ¹³C-terminal residues [36], [37], [38], [39], [40]. The resulting structure is well described as a hydrophobic core with flaring tails [37]. To characterize the dynamical differences between the two conformational states, the incoherent elastic neutron scattering experiments in a wide q range and with the several instrumental energy resolutions were performed. The non-Gaussian behavior in wide q-range was analyzed by the bimodal distribution model. The fraction of the surface flexible part increases by partial unfolding. The difference in dynamical heterogeneity between two different conformational states appeared more distinctly in the dried condition. In contrast, the hydration makes this difference obscure, indicating that the protein dynamics is influenced by hydration. We also found that the apparent dynamical transition depended on the experimentally observable time scale and the time scale dependent dynamics were different between the wild type and the truncated mutant for both the dried and hydrated samples. The hierarchy of the protein dynamics in the time scale is changed by protein folding, and the native and non-native proteins have the different dynamical structures.