Structure and dynamics of oligomeric intermediates in β2-microglobulin self-assembly

Abstract

β(2)-Microglobulin is a 99-residue protein with a propensity to form amyloid-like fibrils in vitro which exhibit distinct morphologies dependent on the solution conditions employed. Here we have used ion mobility spectrometry-mass spectrometry to characterize the oligomeric species detected during the formation of worm-like fibrils of β(2)-microglobulin at pH 3.6. Immediately upon sample dissolution, β(2)-microglobulin monomer and oligomers-the latter ranging in size from dimer to hexamer-are present as a pool of rapidly interconverting species. Increasing the ionic strength of the solution initiates fibril formation without a lag-phase whereupon these oligomers become more stable and higher-order species (7-mer to >14-mer) are observed. The oligomers detected have collision cross-sectional areas consistent with a linearly stacked assembly comprising subunits of native-like volume. The results provide insights into the identity and properties of the transient, oligomeric intermediates formed during assembly of worm-like fibrils and identify species that differ significantly from the oligomers previously characterized during the nucleated assembly of long, straight fibrils. The data presented demonstrate the interrelationship between different fibril-forming pathways and identify their points of divergence.

Figure 1

The formation of worm-like (WL) fibrils of β₂m (0.4 mg mL⁻¹ based on monomer; pH 3.6, 100 mM ammonium acetate; 20°C). β₂m monomer (open circles) was quantified over time by ESI-IMS-MS. The m/z spectra were normalized to the (M+H)⁺ ions of des-Arg bradykinin (1.4 μM, added immediately before analysis) at m/z 904 for monomer quantification (3). (Error bars) Standard deviation of the mean over three replicate experiments. WL fibril formation was monitored simultaneously by thioflavin-T fluorescence (thick solid line). A control in which β₂m (0.4 mg mL⁻¹) was incubated in 10 mM ammonium acetate (pH 3.6; 20°C) was also analyzed; thioflavin-T fluorescence confirmed that fibrils do not form under these conditions (thick shaded line). (Inset) Negative stain EM image of fully formed WL fibrils (the scale bar = 100 nm).

Figure 2

ESI-IMS-MS driftscope plots (drift time versus m/z versus intensity; left) and m/z spectra (right) showing β₂m (0.4 mg mL⁻¹ based on monomer; 20°C; pH 3.6) oligomers detected. (A and B) 10 mM ammonium acetate; (C and D) 100 mM ammonium acetate, after 1 min; (E and F) 100 mM ammonium acetate, after 20 min. The numbers above the peaks indicate the size of each oligomer (e.g., 3 = trimer). The oligomer region (>m/z 6000) in the m/z spectra has been magnified by a factor of 10.

Figure 3

β₂m oligomer population (0.4 mg mL⁻¹ based on monomer) before (t = −10–0 min; 10 mM ammonium acetate; pH 3.6) and during WL fibril formation (t = 0–180 min; 100 mM ammonium acetate, pH 3.6). (Solid circles) β₂m monomer; (open circles) trimer; (solid triangles) hexamer; and (shaded diamonds) 9-mer. For each oligomer, signals were normalized to a value of 1 for the most intense signal detected during the time course. The data are fitted to exponential functions (solid lines). (Thick shaded line) Thioflavin-T fluorescence signal.

Figure 4

β₂m oligomer dynamics change as a function of assembly time. ¹⁴N- and ¹⁵N-labeled β₂m were incubated separately under WL fibril-forming conditions (0.4 mg mL⁻¹ based on monomer; 100 mM ammonium acetate, pH 3.6). The two samples were mixed in an equimolar ratio at various time points and ESI-MS spectra acquired within 1 min of mixing. (A) Predicted m/z spectra for dimer, trimer, tetramer, and pentamer, assuming full ¹⁴N/¹⁵N-labeled subunit exchange has occurred. If subunit exchange with the bulk protein pool is rapid then, in the case of the dimer (n = 2), a 1:2:1 ratio of ¹⁴N/¹⁴N:¹⁴N/¹⁵N:¹⁵N/¹⁵N will be observed. If the exchange is slow or does not occur, then a 1:1 ratio of ¹⁴N/¹⁴N:¹⁵N/¹⁵N will be observed. (B) ESI-MS spectra (m/z 3200–3800) at t = 1, 20, and 180 min. The dimer and the more highly charged trimer (+10, +11) and tetramer (+13) ions exchange rapidly throughout the time course, but are barely populated by 180 min. (C) ESI-MS spectra (m/z 4300–5600) at t = 1, 20, and 180 min. The lower charge-state ions of the trimer (+7, +8), the tetramer (+9, +10, +11) and all the pentamer ions exchange rapidly at t = 1 min but display slow exchange at 180 min, indicating less-dynamic quaternary structures.

Figure 5

ESI-IMS-MS collision cross-sectional areas (Ω; Ų) for β₂m oligomers detected during WL fibril assembly versus molecular mass (kDa). (Solid circles) β₂m oligomers (dimer to 14-mer) populated during WL fibril assembly (100 mM ammonium acetate, pH 3.6). (Solid crosses) β₂m oligomers (dimer to tetramer) populated during LS fibril assembly (100 mM ammonium formate, pH 2.5). (Error bars) One standard deviation in Ω between charge states of a protein. In silico models were constructed using the PyMOL Molecular Graphics System and their Ω-values calculated (34,40). In silico predicted Ω-values are shown for a linear assembly of nativelike β₂m monomers stacked edge-to-edge (open circles), spherical oligomeric structures (open triangles), and an extended model with the N-terminal strand A and the C-terminal strand B of each protein subunit unstructured (open squares). (Solid line) The Ω expected for a protein of a given mass assuming a spherical assembly with a density of 0.44 Da Å⁻³ (31).

Figure 6

ESI-IMS-MS collision cross-sectional areas (Ω; Ų) of individual charge-state ions of β₂m trimer (triangles) and tetramer (circles) arising under long, straight (LS) fibril-forming conditions at pH 2.5 (open symbols) and WL fibril-forming conditions at pH 3.6 (solid symbols). ¹⁴N-labeled β₂m (0.4 mg mL⁻¹ based on monomer; pH 3.6) and ¹⁵N-labeled β₂m (0.4 mg mL⁻¹ based on monomer; pH 2.5) were incubated separately for 20 min before being mixed in an equimolar ratio and analyzed immediately. The trimer ions and the +10 to +13 tetramer ions are common to the oligomers detected on-pathway to both LS and WL fibrils. Only the tetramers detected on-pathway to WL fibrils populate the more compact +7 to +9 charge states.

Figure 7

Compact tetramers divert assembly to WL fibrils at pH 2.5. The LS fibril-assembly of β₂m was initiated (β₂m 0.4 mg mL⁻¹ based on monomer; pH 2.5) and then subjected to increasing buffer concentrations. (A) Thioflavin-T fluorescence analyses showing the spontaneous formation of WL fibrils with >300 mM ammonium formate buffer; (B–D) ESI-IMS-MS driftscope plots showing β₂m oligomers at t = 1 min during fibril assembly with increasing ammonium formate buffer (B, 100 mM; C, 200 mM, and D, 400 mM). The numbers above each peak indicate the oligomer size. The +7 and +8 charge-state ions of the tetramer are highlighted to show their increase in their population with increasing ionic strength.