Kinetic analysis of amyloid protofibril dissociation and volumetric properties of the transition state

Abstract

We present here the first detailed kinetic analysis of the dissociation reaction of amyloid protofibrils by utilizing pressure as an accelerator of the reaction. The experiment is carried out on an excessively diluted typical protofibril solution formed from an intrinsically denatured disulfide-deficient variant of hen lysozyme with Trp fluorescence as the reporter in the pressure range 3-400 MPa. From the analysis of the time-dependent fluorescence decay and the length distribution of the protofibrils measured on atomic force microscopy, we conclude that the protofibril grows or decays by attachment or detachment of a monomer at one end of the protofibril with a monomer dissociation rate independent of the length of the fibril. Furthermore, we find that the dissociation reaction is strongly dependent on pressure, characterized with a negative activation volume DeltaV(odouble dagger) = -50.5 +/- 1.60 ml mol(-1) at 0.1 MPa and with a negative activation compressibility Deltakappa(double dagger) = -0.013 +/- 0.001 ml mol(-1) bar(-1) or -0.9 x 10(-6) ml g(-1) bar(-1). These results indicate that the protofibril is a highly compressible high-volume state, but that it becomes less compressible and less voluminous in the transition state, most probably due to partial hydration of the existing voids. The system eventually reaches the lowest-volume state with full hydration of the monomer in the dissociated state.

FIGURE 1

Pressure-induced changes of Trp fluorescencce spectrum of matured protofibrils of the disulfide-deficient hen lysozyme (0SS) at 350 MPa at 25°C. The protofibril solution (8 mg ml⁻¹) had been prepared by incubating the lyophilized 0SS sample in 20 mM sodium acetate buffer, 30 mM sodium chloride (pH 4.0) for 8 months and was diluted to 30 μg ml⁻¹ in the same buffer just before applying pressure. The top spectrum was measured at 10 min after the pressure jump and the subsequent spectra were measured at 2-min intervals until the bottom spectrum was obtained at 240 min. Note the shift of the maximum emission from 338 to 350 nm concomitant with the decrease in intensity.

FIGURE 2

Plot of the logarithm of normalized fluorescence intensity, ln ((I − I^∞) / (I^o − I^∞)), at 338 nm against time at a constant pressure between 50 and 400 MPa at 25°C (Data taken from Fig. 1). The straight lines are best-fit of the data points to Eq. 1, the slope giving apparent dissociation rate constant (k_obs) at respective pressures.

FIGURE 3

Histogram of the length distribution of amyloid protofibrils of the disulfide-deficient hen lysozyme. The protofibril solution (8 mg ml⁻¹) had been prepared by incubating the lyophilized 0SS in 20 mM sodium acetate buffer, 30 mM sodium chloride, pH 4.0, for 8 months. The length of each protofibril was measured on the atomic force microscopy image over a sufficient number of protofibril fibers. The solid line is a fit of the experimentally determined population C_l of the fibril of length l (nm) with an exponential function of length l, giving C_l ∝ exp(−l / 394).

FIGURE 4

Plot of logarithm of observed dissociation rate (k_obs) (obtained from Fig. 2) against pressure. The original solution of 0SS protofibrils (8 mg ml⁻¹) was excessively diluted to 30 μg ml⁻¹ in 20 mM sodium acetate buffer, 30 mM sodium chloride (pH 4.0) just before applying pressure for irreversible dissociation at 25°C. The solid line represents the least-squares fit of the experimental data to Eq. 3, giving ΔV^0‡ = −50.5 ± 1.6 ml mol monomer⁻¹ and Δκ^‡ = −0.013 ± 0.0007 ml mol monomer⁻¹ bar⁻¹ (1 bar = 0.1 MPa). Extrapolation of the curve to 0.1 MPa gives k_obs = 0.000773 min⁻¹ or τ = 1290 min or 21.6 h.

FIGURE 5

The volume diagram for 0SS in the dissociation reaction of amyloid protofibrils at 0.1 MPa and at 400 MPa at 25°C. At 0.1 MPa, the transition state is ∼50 ml mol⁻¹ below the protofibril state (ΔV°^‡ = −50.5 ± 1.6 ml·mol monomer⁻¹). However, this difference becomes almost null at 400 MPa because the compressibility is larger in the protofibril state than in the monomeric state (Δκ^‡ < 0). Note that the volume changes between 0.1 MPa and 400 MPa are unknown for both the protofibril and monomeric states so that the relative levels between the two pressures are arbitrarily drawn.