Effects of pressure on the dynamics of an oligomeric protein from deep-sea hyperthermophile

Abstract

Inorganic pyrophosphatase (IPPase) from Thermococcus thioreducens is a large oligomeric protein derived from a hyperthermophilic microorganism that is found near hydrothermal vents deep under the sea, where the pressure is up to 100 MPa (1 kbar). It has attracted great interest in biophysical research because of its high activity under extreme conditions in the seabed. In this study, we use the quasielastic neutron scattering (QENS) technique to investigate the effects of pressure on the conformational flexibility and relaxation dynamics of IPPase over a wide temperature range. The β-relaxation dynamics of proteins was studied in the time ranges from 2 to 25 ps, and from 100 ps to 2 ns, using two spectrometers. Our results indicate that, under a pressure of 100 MPa, close to that of the native environment deep under the sea, IPPase displays much faster relaxation dynamics than a mesophilic model protein, hen egg white lysozyme (HEWL), at all measured temperatures, opposite to what we observed previously under ambient pressure. This contradictory observation provides evidence that the protein energy landscape is distorted by high pressure, which is significantly different for hyperthermophilic (IPPase) and mesophilic (HEWL) proteins. We further derive from our observations a schematic denaturation phase diagram together with energy landscapes for the two very different proteins, which can be used as a general picture to understand the dynamical properties of thermophilic proteins under pressure.protein dynamics | energy landscape | denaturation phase diagram | quasielastic neutron scattering | mode coupling theory T he biological functions of proteins, such as enzyme catalysis, are often understood from their crystallographic structures (1). On the other hand, it is crucial to take into account dynamic behavior to fully comprehend these functions (2, 3). In vivo, proteins are in constant motion among different conformations (3-5). The thermal energy, which is of the order of k B T per atom, where k B is the Boltzmann constant and T is the absolute temperature, triggers biomolecules to sample different conformations around the average structure. These conformations are also known as conformational substates (CSs) (5). Fluctuations among these CSs play an important role in protein function (3,5). These lead to the concept of a multidimensional potential energy landscape (EL) that specifies a complete description of CSs in proteins (6-9). The existence of an EL was proposed by H. Frauenfelder and others in the 1970s and has been validated both by computations and by experiments (6-12).Proteins show various dynamic phenomena over a wide range of timescales, from picoseconds to milliseconds (13). A fast dynamic process, on a timescale of a picosecond to 10 ns, also known as β-relaxation, occurs due to small amplitude fluctuations in atoms/ molecules, such as loop motions and side-chain rotations (14). The energy barrier or activation energy (E A ) between different CSs for this process is smaller than k B T (15). On t...

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