Enhanced accessibility and hydrophobicity of amyloidogenic intermediates of the β2-microglobulin D76N mutant revealed by high-pressure experiments

Abstract

β2-Microglobulin (β2m) is the causative protein of dialysis-related amyloidosis. Its unfolding mainly proceeds along the pathway of N_C →U_C ⇄ U_T, whereas refolding follows the U_T → I_T (→N_T) →N_C pathway, in which N, I, and U are the native, intermediate, and unfolded states, respectively, with the Pro32 peptidyl-prolyl bond in cis or trans conformation as indicated by the subscript. It is noted that the I_T state is a putative amyloidogenic precursor state. Several aggregation-prone variants of β2m have been reported to date. One of these variants is D76N β2m, which is a naturally occurring amyloidogenic mutant. To elucidate the molecular mechanisms contributing to the enhanced amyloidogenicity of the mutant, we investigated the equilibrium and kinetic transitions of pressure-induced folding/unfolding equilibria in the wild type and D76N mutant by monitoring intrinsic tryptophan and 1-anilino-8-naphthalene sulfonate fluorescence. An analysis of kinetic data revealed that the different folding/unfolding behaviors of the wild type and D76N mutant were due to differences in the activation energy between the unfolded and the intermediate states as well as stability of the native state, leading to more rapid accumulation of I_T state for D76N in the refolding process. In addition, the I_T state was found to assume more hydrophobic nature. These changes induced the enhanced amyloidogenicity of the D76N mutant and the distinct pathogenic symptoms of patients. Our results suggest that the stabilization of the native state will be an effective approach for suppressing amyloid fibril formation of this mutant.

Keywords: amyloid; biophysics; fluorescence; high-pressure experiment; mutant; protein folding; β2-microglobulin.

Figure 1

Crystal structures ofβ2m(PDB ID: 2yxf (1)). The side chains of P32, D76, C25, and C80 are depicted as balls and sticks.

Figure 2

Equilibrium and kinetic measurements of pressure-induced unfolding.A, pressure-dependent spectral changes in the tryptophan fluorescence of wild-type β2m. The blue and red lines indicate the spectra obtained at 5 and 450 MPa, respectively. These spectra were recorded 30 min after pressure was changed to the respective points. B, pressure dependence of the fraction of the native state based on the <ν> values for the wild type (red) and D76N (green). The broken lines are the theoretical curves based on the two-state unfolding model expressed by Equations 1 and 2 (see main text). C and D, the unfolding kinetics of wild-type (C) and D76N (D) β2ms probed through the time-dependent <ν> value. The continuous lines are single exponential curves fit to the data. The dotted lines indicate the <ν> value of the N state at 5 MPa before the unfolding reaction. E, plots of unfolding rate constants against the respective pressures for the wild type (red) and D76N (green).

Figure 3

Kinetic measurements of refolding from the pressure-induced unfolded state.A, ANS fluorescence spectra of the pressure-induced unfolded state (blue) and refolded state from the unfolded state (black) of the wild type are shown. B and C, refolding kinetics monitored by ANS fluorescence for the wild type (B) and D76N (C) are shown, where intensity was normalized with respect to the initial (unfolded state) intensity of the ANS fluorescence. The white lines are single exponential curves fit for rate constants. D, the pressure-dependent rate constants obtained for the wild type (red) and D76N (green) are shown.

Figure 4

Model fitting of experimental data.A, schematic presentation of the proposed four-state model. B–D, comparison of theoretical curves and experimental data for WT (red) and D76N (green). B, pressure dependence of the fraction of the native state. C, plots of refolding (open circle) and unfolding (solid circle) rate constants against the respective pressures. D, the pressure-dependent final intensity of ANS fluorescence (open circle) and exponential amplitude (solid triangle) obtained from the refolding experiments are shown. In panels B–D, the continuous lines are theoretical curves derived from the global fitting of the equilibrium (B) and kinetic (C and D) data to the four-state model (see Experimental procedures). Thus, although the data in B is the same as those in Figure 2B, the theoretical curve is different from that in Figure 2B. E and F, ΔG₀ and ΔV diagrams obtained for WT (red) and D76N (green). G–J, calculated time-dependent populations of each species during refolding upon 400→5 MPa change for WT (G) and D76N (H), and those upon 400→100 MPa change for WT (I) and D76N (J).

Figure 5

Representation of species appearing in the folding process of β2m at ambient pressure (A) and at moderate pressure (100 MPa) (B). The descriptions in green in (A) indicate the effects of the D76N mutation. The descriptions in red indicate the effects of pressure. The red arrows indicate the rate-limiting steps at respective pressures. The broken lines indicate the microscopic steps, which associated with the observable slow phases at respective pressures. Especially, the kinetic coupling with both the first and second steps occurred at around 100 MPa (B).