Serum amyloid A 2.2 refolds into a octameric oligomer that slowly converts to a more stable hexamer

Abstract

Serum amyloid A (SAA) is an inflammatory protein predominantly bound to high-density lipoprotein in plasma and presumed to play various biological and pathological roles. We previously found that the murine isoform SAA2.2 exists in aqueous solution as a marginally stable hexamer at 4-20°C, but becomes an intrinsically disordered protein at 37°C. Here we show that when urea-denatured SAA2.2 is dialyzed into buffer (pH 8.0, 4°C), it refolds mostly into an octameric species. The octamer transitions to the hexameric structure upon incubation from days to weeks at 4°C, depending on the SAA2.2 concentration. Thermal denaturation of the octamer and hexamer monitored by circular dichroism showed that the octamer is ∼10°C less stable, with a denaturation mid point of ∼22°C. Thus, SAA2.2 becomes kinetically trapped by refolding into a less stable, but more kinetically accessible octameric species. The ability of SAA2.2 to form different oligomeric species in vitro along with its marginal stability, suggest that the structure of SAA might be modulated in vivo to form different biologically relevant species.

Fig. 1

Refolding of SAA2.2 monitored by size-exclusion chromatography (SEC) (A), and analytical ultracentrifugation (AUC) sedimentation equilibrium (SE) analysis of “mature” (B) and “fresh” (C) SAA2.2. Denatured SAA2.2 (0.8 mg/mL, 20 mM Tris, 150 mM NaCl, 6M Urea, pH 8.2) was dialyzed against Tris buffer (20mM Tris, pH 8.2) at 4°C and its oligomeric structure was followed by SEC for 1 month. SAA2.2 refolds initially to an octameric species that later rearranges to a hexamer (A). Representative AUC-SE traces at 10,000 rpm (○), 12,000 rpm (□) and 19,000 rpm (△) for SAA2.2 (20 mM Tris pH 8.2, 150 mM NaCl, 4°C) are shown, where the solid lines represent the global fit of mature (B) and fresh (C) SAA2.2 according to equation 1. The lower panel shows the residuals for each fit. The molecular weight (MW) obtained from the fit for the “mature” and “fresh” protein was 70.5± 0.6 kDa and 85.1± 0.5 kDa, respectively.

Fig. 2

CD secondary structure characterization (A) and thermal denaturation (B) of SAA2.2 hexamer and octamer. Far-UV CD spectrums of SAA2.2 hexamer (■) and octamer (◆) at 4°C show that they have similar secondary structure. Thermal denaturation curves of SAA2.2 hexamer (■) and octamer (◆) monitored by far-UV CD indicate that the octamer is ~10 °C less stable than the hexamer. Samples contained 0.1 mg/mL SAA2.2 in 20 mM phosphate buffer.

Fig. 3

Refolding of SAA2.2 (0.8mg/ml in 20mM Tris, 6M Urea, 150mM NaCl, pH 8.2) was performed at room temperature (RT) against Tris buffer (20 mM Tris, pH 8.2). After ~12h ~25μl of the sample was loaded in an analytical gel filtration column to determine the oligomeric state of SAA2.2. A sample from the same SAA2.2 stock was refolded at 4°C as a control, and formed mostly an octamer species.

Fig. 4

Concentration dependence conversion of SAA2.2 octamer to hexamer. SAA2.2 was purified and dissolved in buffer A (20mM Tris, 6M Urea, 400mM NaCl, pH 8.) and aliquoted at three different concentrations (A: 0.25mg/ml, B: 0.8mg/ml and C: 1.8mg/ml) and allowed to refold at 4°C in for 14hrs in 500 – 1000X refolding buffer (20mM Tris, pH 8). SAA was aliquoted in sterile tubes and stored under refrigerated conditions. At regular intervals (as specified in the figure) samples were analyzed on a Superdex 200® analytical gel filtration column.