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, 112 (45), 13886-91

Effects of Pressure on the Dynamics of an Oligomeric Protein From Deep-Sea Hyperthermophile

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Effects of Pressure on the Dynamics of an Oligomeric Protein From Deep-Sea Hyperthermophile

Utsab R Shrestha et al. Proc Natl Acad Sci U S A.

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.

Keywords: denaturation phase diagram; energy landscape; mode coupling theory; protein dynamics; quasielastic neutron scattering.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. S1.
Fig. S1.
Enzymatic activities for Tt-IPPase (41) and HEWL (45) as functions of temperature. The optimal temperatures for activity are 323 K (50 °C) and 358 K (85 °C) for HEWL and IPPase, respectively.
Fig. 1.
Fig. 1.
Normalized QENS spectra from protein samples and data fitting. (A and B) Spectra measured at DCS from IPPase and HEWL, respectively, at Q = 0.8 Å−1 for temperatures from 298 to 363 K along with resolution. (C and D) DCS data fitted in energy domain for IPPase and HEWL, respectively, at Q = 0.8 Å−1 and T = 363 K. (E and F) Spectra measured at HFBS from IPPase and HEWL, respectively, at Q = 0.9 Å−1 for temperatures from 298 to 363 K along with resolution. (G and H) HFBS data fitted in energy domain for IPPase and HEWL, respectively, at Q = 0.9 Å−1 and T = 363 K. The background is fitted linearly, and elastic and quasielastic components are fitted with delta and Lorentzian functions, respectively. In this figure, and in subsequent figures, error bars represent ±1 SD.
Fig. S2.
Fig. S2.
Lorentzian half width at half maximum (HWHM) of IPPase and HEWL from DCS (A and B) and HFBS (C and D), respectively. Error bars represent 1 SD.
Fig. 2.
Fig. 2.
Analysis of QENS data in the energy domain at all measured temperatures. (A and B) Elastic incoherent structure factor (EISF) for IPPase and HEWL, calculated from the data measured at DCS. (C and D) EISFs calculated from the data obtained at HFBS. (E and F) Fraction of mobile H atoms in a confined diffusion sphere (1 − p0) as a function of temperature for IPPase (yellow circles) and HEWL (blue spheres), calculated from the data obtained at DCS and HFBS, respectively.
Fig. S3.
Fig. S3.
Elastic incoherent structure factor (EISF) calculated using Eq. S8 to validate those as calculated in Fig. 2 in the main text using Eq. S1 in energy domain. (A and B) EISF for IPPase and HEWL, calculated from I(Q, t ∼ 25 ps) of DCS results. (C and D) EISFs calculated from I(Q, t ∼ 2 ns) of HFBS results. The solid lines correspond to the fitting of EISF described in the main text.
Fig. 3.
Fig. 3.
Intermediate scattering function (ISF) calculated from DCS and HFBS spectra. (A–F) ISFs of H atoms in hydrated IPPase (A, C, and E) and HEWL (B, D, and F), respectively, calculated from DCS data. (G–L) ISFs of H atoms in hydrated IPPase (G, I, and K) and HEWL (H, J, and L), respectively, calculated from HFBS data. Here, we show results at three temperatures: T = 298, 338, and 363 K. ISFs are calculated at a series of Q values from 0.5 to 1.8 Å−1. Solid lines represent the curves fitted by Eq. 1.
Fig. 4.
Fig. 4.
Fitting parameters obtained from the MCT analysis of the ISF from DCS data and mean-squared displacement (MSD) of IPPase and HEWL from HFBS data. (A and B) First-order logarithmic decay parameter H1(Q, T) as a function of Q for IPPase and HEWL, respectively. (Inset) B1(T) as a function of temperature for IPPase and HEWL. (C) β-Relaxation time constant, τβ(T) plotted as a function of temperature. Dashed lines represent Arrhenius fit of the relaxation time τβ for IPPase and HEWL. (D) MSD (<x2>) of H atoms in protein samples, IPPase and HEWL, measured by elastic incoherent neutron scattering at HFBS. The dynamic transition temperature (TD) for IPPase and HEWL are observed around 220–240 K.
Fig. 5.
Fig. 5.
Schematic picture of phase diagram and energy landscape in IPPase and HEWL under high pressure and temperature. (Left) Denaturation phase diagram of IPPase and HEWL (shaded region) as functions of temperature and pressure. The axes in the diagram are not drawn to scale. (Right) Schematic plot of cross-sections through a highly simplified energy landscape of atomic fluctuations for different conformational substates (CSs) in IPPase and HEWL under ambient and 100 MPa (1 kbar) of pressure.

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