A measure of conformational entropy change during thermal protein unfolding using neutron spectroscopy

Abstract

Thermal unfolding of proteins at high temperatures is caused by a strong increase of the entropy change which lowers Gibbs free energy change of the unfolding transition (DeltaG(unf) = DeltaH - TDeltaS). The main contributions to entropy are the conformational entropy of the polypeptide chain itself and ordering of water molecules around hydrophobic side chains of the protein. To elucidate the role of conformational entropy upon thermal unfolding in more detail, conformational dynamics in the time regime of picoseconds was investigated with neutron spectroscopy. Confined internal structural fluctuations were analyzed for alpha-amylase in the folded and the unfolded state as a function of temperature. A strong difference in structural fluctuations between the folded and the unfolded state was observed at 30 degrees C, which increased even more with rising temperatures. A simple analytical model was used to quantify the differences of the conformational space explored by the observed protein dynamics for the folded and unfolded state. Conformational entropy changes, calculated on the basis of the applied model, show a significant increase upon heating. In contrast to indirect estimates, which proposed a temperature independent conformational entropy change, the measurements presented here, demonstrated that the conformational entropy change increases with rising temperature and therefore contributes to thermal unfolding.

FIGURE 1

The stability curve of α-amylase from B. licheniformis (BLA) with ΔG_unf as function of temperature. Here the Gibbs free energy for the unfolding transition is given by ΔG_unf(T) = G^U − G^F = ΔH(T) − T × ΔS(T) = ΔH_m (1 − T/T_m) + Δc_p [T − T_m − T × ln(T/T_m)] with the melting temperature T_m = 103°C, the enthalpy change ΔH_m = 2686 kJ mol⁻¹, and the change in heat capacity Δc_p = 32.74 kJ mol⁻¹ K⁻¹ (Fitter, unpublished results; Feller et al., 1999).

FIGURE 2

Difference spectra of BLA in the folded (a) and the unfolded (b) state. The number of scattered neutrons is given as a function of energy transfer (here for sample at T = 30°C and Q = 1.6 Å⁻¹). The experimental data (symbols) were fitted by a total scattering function (thick solid line) which includes elastic scattering having (small) energy transfer values which cannot be resolved by the resolution function (Γ_res = 120 μeV (fwhm); dashed line), quasielastic scattering (shaded area) which is described by a Lorentzian (width = 150 ± 10 μev (hwhm)), and a constant background (not shown here). The lower part (c) of this figure shows the Q dependence of A₀ (symbols) and the fitted Q dependence according to the diffusion inside a sphere model (solid lines).

FIGURE 3

A comparison of spectra from BLA in D₂O buffer solution (folded state, dashed-dotted line; unfolded state, solid line) and from D₂O buffer solely (dashed line). All spectra were measured at scattering angles between 60° and 65°, which corresponds to an average Q value of 1.27 Å⁻¹. Panels a–c show the increase of quasielastic scattering with rising temperature, which is more pronounced for the unfolded state as compared to the folded state.

FIGURE 4

Elastic incoherent structure factors (A₀) determined from difference spectra (see Fig. 2) given for the folded state (a) and the unfolded state (b) at three different temperatures. The statistical error of the obtained A₀ (symbols, see also Fig. 2 c) is about ±4%. The solid lines represent structure factors calculated on the basis of the diffusion inside a sphere model. The corresponding model parameters (radius of the sphere) are given in Table 1.