The role of conformational flexibility in β2-microglobulin amyloid fibril formation at neutral pH

Abstract

Rationale: Amyloid formation is implicated in a number of human diseases. β(2)-Microglobulin (β(2)m) is the precursor protein in dialysis-related amyloidosis and it has been shown that partial, or more complete, unfolding is key to amyloid fibril formation in this pathology. Here the relationship between conformational flexibility and β(2)m amyloid formation at physiological pH has been investigated.

Methods: HDX-ESI-MS was used to study the conformational dynamics of β(2)m. Protein engineering, or the addition of Cu(2+) ions, sodium dodecyl sulphate, trifluoroethanol, heparin, or protein stabilisers, was employed to perturb the conformational dynamics of β(2)m. The fibril-forming propensities of the protein variants and the wild-type protein in the presence of additives, which resulted in >5-fold increase in the EX1 rate of HDX, were investigated further.

Results: ESI-MS revealed that HDX occurs via a mixed EX1/EX2 mechanism under all conditions. Urea denaturation and tryptophan fluorescence indicated that EX1 exchange occurred from a globally unfolded state in wild-type β(2)m. Although >30-fold increase in the HDX exchange rate was observed both for the protein variants and for the wild-type protein in the presence of specific additives, large increases in exchange rate did not necessarily result in extensive de novo fibril formation.

Conclusions: The conformational dynamics measured by the EX1 rate of HDX do not predict the ability of β(2)m to form amyloid fibrils de novo at neutral pH. This suggests that the formation of amyloid fibrils from β(2)m at neutral pH is dependent on the generation of one or more specific aggregation-competent species which facilitate self-assembly.

Figure 1

The solution structure (PDB accession code 1JNJ) of wild-type human β₂m. The residues/regions mutated in the four variants in these studies are highlighted: ΔN6 (green), I7A (dark blue), P32G (cyan) and V37A (pink). The position of the FG loop that differs markedly in sequence between human and murine β₂m is shown in black. The β-strands are annotated A–G and the disulphide bond is shown as an orange stick linking strands B and F.

Figure 2

HDX-ESI-MS of wild-type β₂m. (A) D→H exchange of wild-type β₂m (pH 7.0, 37 °C). Protein molecules which have undergone exchange via an EX2 mechanism only are shown on the right in red, while those which have undergone exchange via both an EX2 and EX1 mechanism are shown on the left in blue. (B) Plot showing the intensity of the EX1 peak relative to the total protein signal over time. The data are fitted to a single exponential (red line).

Figure 3

Determination of wild-type β₂m unfolding rate. (A) Plot showing the unfolding of wild-type human β₂m measured using tryptophan fluorescence (pH 7.0, 37 °C) at increasing urea concentration (4.95-7.65 M urea in 0.45 M increments). The data are shown in grey and a single exponential fit to the data is shown as a black line. (B) Plot of the natural logarithm of the unfolding rate constant of wild-type human β₂m (pH 7.0, 37 °C) vs. the urea concentration (M). The data are shown as grey squares and the linear fit to the data in black. The error on each point (2 standard deviations of the mean) is smaller than the data marker.

Figure 4

HDX-ESI-MS of wild-type β₂m in presence of Cu²⁺, TFE or SDS and also of ΔN6 β₂m. D→H exchange of wild-type human β₂m (pH 7.0, 37 °C) in the presence of (A) a 2-fold molar excess of Cu²⁺ ions; (B) 10 % (v/v) TFE; and (C) 1 mM SDS. (D) D→H exchange of ΔN6 β₂m (pH 7.0, 22 °C). In each case the left-hand image shows the mass profile of the ESI-MS spectrum. Protein molecules which have undergone exchange via an EX2 mechanism only are shown on the right in red, while peaks that arise from molecules which have undergone exchange via both EX2 and EX1 mechanisms are shown on the left in blue. Peaks in (A) and (D) at higher mass (grey) result from potassium and sodium adducts. The right-hand column shows plots of EX1 peak intensity relative to the total protein signal over time. The data are fitted to a single exponential (red line) in each case.

Figure 5

The EX1 rate of HDX of wild-type human β₂m, its variants and murine β₂m in the presence of a range of different solution conditions. All reactions were measured at pH 7.0, 37 °C, except for those marked with * which were carried out at 22 °C. The error bars depict two standard deviations of the mean. The dashed line denotes the threshold of a rate of HDX ≥ 5–fold that of wild-type human β₂m.

Figure 6

Fibril growth assays at pH 7.0, 37 °C with shaking at 200 rpm monitored by thioflavin-T fluorescence. A (i) wild-type β₂m in the presence of 2 mM SDS (blue) or 1 mM SDS (red). Negative stain EM images of the fibrils resulting from incubation with (ii) 2 mM SDS and (iii) 1 mM SDS for 14 days are shown. B (i) wild-type human β₂m in the presence of 20 % (v/v) TFE; (ii) the reaction end-point (14 days) observed by EM is shown. C (i) ΔN6 β₂m; (ii) the reaction end-point (14 days) observed by EM is shown. D (i) I7A β₂m; (ii) the reaction end-point (14 days) observed by EM is shown. E (i) I7A β₂m; (ii) the reaction end-point (14 days) observed by EM is shown. Scale bars are shown in red and indicate 500 nm.