High-pressure dynamics of hydrated protein in bioprotective trehalose environment

We present a pressure-dependence study of the dynamics of lysozyme protein powder immersed in deuterated $\alpha ,\alpha$-trehalose environment via quasielastic neutron scattering (QENS). The goal is to assess the baroprotective benefits of trehalose on biomolecules by comparing the findings with those of a trehalose-free reference study. While the mean-square displacement of the trehalose-free protein (hydrated to $d_{{\mathrm{D}}_2\mathrm{O}}\simeq 40$ w%) as a whole, is reduced by increasing pressure, the actual observable relaxation dynamics in the picoseconds to nanoseconds time range remains largely unaffected by pressure—up to the maximum investigated pressure of 2.78(2) Kbar. Our observation is independent of whether or not the protein is mixed with the deuterated sugar. This suggests that the hydrated protein's conformational states at atmospheric pressure remain unaltered by hydrostatic pressures, below 2.78 Kbar. We also found the QENS response to be totally recoverable after ambient pressure conditions are restored. Small-angle neutron diffraction measurements confirm that the protein-protein correlation remains undisturbed. We observe, however, a clear narrowing of the QENS response as the temperature is decreased from 290 to 230 K in both cases, which we parametrize using the Kohlrausch-Williams-Watts stretched exponential model. Only the fraction of protons that are immobile on the accessible time window of the instrument, referred to as the elastic incoherent structure factor, is observably sensitive to pressure, increasing only marginally but systematically with increasing pressure.

Figure 1

Representative raw quasielastic neutron (QENS) response collected on the BASIS spectrometer at temperature $T=290$ K and wavevector $Q=0.5$ $\AA {}^{-1}$. The black circles show the total signal from the ${\mathrm{D}}_2\mathrm{O}$-hydrated lysozyme powder in the Al container. The black solid line represents the signal from the empty high-pressure Al cell and the blue dashed line shows the instrument resolution function, measured at 100 K using the same ${\mathrm{D}}_2\mathrm{O}$-hydrated sample.

Figure 2

Temperature dependence of the net QENS response from ${\mathrm{D}}_2\mathrm{O}$-hydrated lysozyme after subtraction of container contribution and vanadium normalization at $Q=0.5$ $\AA {}^{-1}$ (left panel) and $Q=1.5$ $\AA {}^{-1}$ (right panel). Data at 100 K was used as a reference resolution function to determine the characteristic relaxation parameters at the higher temperatures.

Figure 3

Evolution of the mean-square displacement (MSD) of hydrogens with motion faster than $\sim 1$ nanosecond, in ${\mathrm{D}}_2\mathrm{O}$-hydrated lysozyme powder with pressure. The harmonic behavior observed at low temperatures changes slope around 220 K. This anharmonicity at the high temperature goes down with increasing pressure. The data was calibrated with the reference MSD value $\langle u_0^2\left(T_0\right)\rangle$ at $T_0=150$ K.

Figure 4

Variation of the stretching exponent parameter ${\beta }_Q$ with momentum transfer $Q$ at temperature $T=290$ K. Solid circles are the observed values at ambient pressure, and the open circles at $P=2.78$ Kbar. The solid and dashed lines are the average values over all $Q$.

Figure 5

Net observed signal (background subtracted) from the ${\mathrm{D}}_2\mathrm{O}$-hydrated protein compound without trehalose (black squares) and with trehalose (red circles). Solid black line shows the corresponding resolution function. The blue lines are the resulting fits to the data using a Kohlrausch-Williams-Watts (KWW) model, discussed in the text.

Figure 6

Pressure dependence of the net response from ${\mathrm{D}}_2\mathrm{O}$-hydrated lysozyme at selected $Q=0.5$ $\AA {}^{-1}$ (left panel) and $Q=0.9$ $\AA {}^{-1}$ (right panel) at 290 K. Black circles are the data at ambient pressure (1 bar) and red circles at 2.78 Kbar. The blue lines are the fits to the data using a Kohlrausch-Williams-Watts (KWW) model, as discussed in the text.

Figure 7

Inverse ${\tau }_Q$ as a function of $Q^2$ for lysozyme at ambient pressure (left panel) and elevated pressure (right panel). Solid lines, denoted Fit 1, are fits of Eq. (3) to the observed values. Dashed lines (Fit 2) are fits of ${\tau }^{-1}\left(Q\right)=D_r{Q}^2/(1+D_r{\tau }_0{Q}^2)$ to the data.

Figure 8

Inverse ${\tau }_Q$ as a function of $Q^2$ for lysozyme and deuterated trehalose compound at selected pressures: ambient pressure (left panel) and elevated pressure (right panel). Labels are the same as described in the legend of Fig. 7

Figure 9

Influence of pressure on the incoherent structure factor [EISF or $x\left(Q\right)$] of lysozyme at 290 K. Symbols represent results obtained, respectively, at ambient pressure (black squares), 1.00 Kbar (green circles), 1.58 Kbar (blue diamonds), and 2.78 Kbar (magenta triangles). Dashed lines are model fits to the data.

Figure 10

Model fits to the elastic incoherent structure factor [EISF or $x\left(Q\right)$] of ${\mathrm{D}}_2\mathrm{O}$-hydrated protein at 290 K: black circles (experimental data); black short-dashed line (three-site jumps model); red long-dashed line [Eq. (4) with confining radius $a_0$ for methyls group allowed to vary]; blue solid line [Eq. (4) with $a_0$ fixed to 1.1 Å, as observed previously 41].

Figure 11

Temperature dependence of the elastic incoherent structure factor [EISF or $x\left(Q\right)$] of lysozyme without trehalose: red squares (290 K), green circles (260 K), and blue triangles (230 K). The left panel shows the values at ambient pressure and the right panel indicates those at 2.78 Kbar. The dashed lines are fits of Eq. (4) with $a_0$ set to 1.1 Å.

Figure 12

Temperature dependence of the elastic incoherent structure factor [EISF or $x\left(Q\right)$] of lysozyme in deuterated trehalose environment.

Figure 13

Influence of pressure on the observed protein-protein peak in hydrated lysozyme at 290 K. Data were taken in situ at BASIS using new diffraction detectors. Symbols represent results obtained, respectively, at ambient pressure (black squares), 1.00 Kbar (green circles), 1.58 Kbar (blue diamonds), and 2.78 Kbar (magenta triangles).