Influence of hydration on the dynamics of lysozyme

Abstract

Quasielastic neutron and light-scattering techniques along with molecular dynamics simulations were employed to study the influence of hydration on the internal dynamics of lysozyme. We identified three major relaxation processes that contribute to the observed dynamics in the picosecond to nanosecond time range: 1), fluctuations of methyl groups; 2), fast picosecond relaxation; and 3), a slow relaxation process. A low-temperature onset of anharmonicity at T approximately 100 K is ascribed to methyl-group dynamics that is not sensitive to hydration level. The increase of hydration level seems to first increase the fast relaxation process and then activate the slow relaxation process at h approximately 0.2. The quasielastic scattering intensity associated with the slow process increases sharply with an increase of hydration to above h approximately 0.2. Activation of the slow process is responsible for the dynamical transition at T approximately 200 K. The dependence of the slow process on hydration correlates with the hydration dependence of the enzymatic activity of lysozyme, whereas the dependence of the fast process seems to correlate with the hydration dependence of hydrogen exchange of lysozyme.

FIGURE 1

Ln(I_el(Q,T)/I_el(Q,10 K)) of dry lysozyme versus Q². The dashed lines represent linear fits using the broad Q range (up to Q² ∼ 3 Å⁻²), whereas the solid lines represent linear fits in the narrower Q range up to Q² ∼ 1 Å⁻².

FIGURE 2

Temperature variations of mean-squared atomic displacement, 〈r²(T)〉. (A) Experimental data at different hydration levels: 0.05 (dry), 0.18, 0.30, and 0.45 h. Crystallization of D₂O prevented accurate measurements of 〈r²(T)〉 in samples at h > 0.45. (B) Simulations for wet lysozyme (0.43 h).

FIGURE 3

Low-temperature behavior of 〈r²(T)〉. (A) Experimental data showing the onset of anharmonicity at T ∼ 120 K. (B) Simulations for wet lysozyme (0.43 h), contributions of methyl and nonmethyl atoms are presented separately. The lines are the extrapolation of low-temperature harmonic behavior.

FIGURE 4

(A) High-frequency dynamic structure factor, S_TOF(Q,ν), summed over all Q. Samples are 0.05 (dry), 0.18, 0.30, 0.50, and 0.80 h. (B) Light-scattering intensity, I(ν). Samples are 0.03 (dry), 0.10, 0.15, 0.20, 0.35, 0.50, 0.75, and 0.85 h. All neutron- and light-scattering spectra were obtained at T = 295 K and normalized at a high-frequency region that is not sensitive to hydration, ∼2.5 THz (∼10 meV).

FIGURE 5

Temperature variations of elastic intensity, I_el(Q,T), in dry sample (0.05 h) corrected for the Debye-Waller factor (○). Solid lines represent the results of the fit using Eq. 2 with a single barrier E ∼ 16 kJ/mol (thin line) and a Gaussian distribution of the energy barriers with E₀ ∼ 16.6 kJ/mol and ΔE ∼ 5.8 kJ/mol (thick line) for the methyl-group rotation.

FIGURE 6

EISF(Q) for the dry sample (0.05 h) at T = 295 K obtained using single Lorentzian approximation for QES (•) and using distribution of energy barriers (○). Solid lines represent the fit to Eq. 4.

FIGURE 7

Distribution of effective rotational correlation times (τ) for methyl groups in lysozyme obtained from molecular dynamics simulations for wet sample (0.43 h) at T = 295 K, (A) presented as a histogram with residue names marked on top, and (B) presented for each residue separately.

FIGURE 8

Neutron scattering susceptibility, χ″_TOF(ν) (symbols), and light-scattering susceptibility, χ″_LS(ν) (solid lines), of samples at different hydrations (neutron-scattering measurements: h ∼0.05 (dry), 0.18, 0.30, 0.50, and 0.80; light-scattering measurements: h ∼0.03 (dry), 0.20, 0.35, 0.50, and 0.85) at T = 295 K. The dashed lines show the slope of high-frequency tail of slow relaxation process (χ″(ν) ∝ ν ^−0.2) and the slope of low-frequency tail of the fast relaxation process (χ″(ν) ∝ ν^0.55).

FIGURE 9

(A) Neutron-scattering susceptibility, χ″_HFBS(ν) and χ"_TOF(ν) of (h ∼ 0.05 (dry), 0.30, and 0.80) at T = 295 K in a broad frequency range. (B) Neutron- (□) and light-scattering (thin solid line) susceptibility spectra of dry samples (h ∼ 0.05 and 0.03) at T = 295 K. The thick solid line shows the fit of the low-frequency spectrum using Eq. 3 (Fig. 6); the dashed line shows estimated methyl-group contribution. High-frequency neutron-scattering spectrum (▵) after correction for the methyl-group contribution agrees well with the light-scattering spectrum.

FIGURE 10

EISF_{hydrated,slow}(Q) of hydrated samples (for definition, see text, Eq. 8) at h ∼ 0.18 (○); 0.30 (▴); 0.50 (∇); 0.80 (♦) at T = 295 K. The dashed lines present fit to the two-site (Eq. 9) and three-site (Eq. 4) jump models (fits are indistinguishable at this Q-range) and the solid lines present fits to freely diffusive motions in a sphere model (Eq. 10).

FIGURE 11

(A) Amplitude of motions (jump distance d and radius of sphere a) involved in slow relaxation process at different hydration levels obtained from the fit to two-site jump model (▴) and to freely diffusive motions in a sphere model (▪). The dashed line shows the jump distance expected for methyl groups. (B) Variation of mobile fraction of hydrogen atoms involved in the slow relaxation process, P_slow, with hydration obtained from the fit to two-site jump model (▴, outside labels) and to freely diffusive motions in a sphere model (□, inside labels). QES intensity integrated in the frequency range from 2 to 8 GHz (○) is presented for a comparison.

FIGURE 12

High-frequency χ″(ν), corrected for the contribution of the slow relaxation process at T = 295 K. (A) Light-scattering and (B) neutron-scattering measurements.

FIGURE 13

Light (□) and neutron (▵) QES intensity corrected for the slow relaxation process and integrated in the frequency range from 50 to 100 GHz as a function of hydration. It reflects essentially the dependence of the fast relaxation process on hydration.

FIGURE 14

(A) Parallel comparison of hydration dependences of the mobile fraction of hydrogen atoms involved in the slow relaxation process (▴) and enzymatic reaction rate, v₀, of the lysozyme to hexasaccharide of N-acetylglucosamine ((GlcNAc)₆) (⋆) as estimated by Rupley and colleagues (5,74). (B) Parallel comparison of hydration dependences of the integrated QES intensity of the fast process (▪) and hydrogen exchange rate (▹) in units of moles of exchanged H atoms/1 mol of lysozyme/24 h as estimated in Schinkel et al. (36). (T = 295 K).