Trans-Suppression of Misfolding in an Amyloid Disease Per Hammarström, Frank Schneider, and Jeffery W. Kelly

Equilibrium analytical ultracentrifugation analysis on the mixture of V30M/FT₂-T119M TTR produced by co-expression demonstrated that tetramers are produced. The binding of the retinol binding protein•vitamin A complex and thyroxine to the mixture of 5 TTR tetramers revealed that the function of the protein and by extension its structure is normal. Retinol binding protein (RBP) binding to TTR was probed by sedimentation equilibrium analysis using a 0.4 mg/mL solution of tetramers 1-5 and RBP (1). Thyroid hormone (T4) binding to a solution of tetramers 1-5 was evaluated by I¹²⁵ labeled T4 using an equilibrium dialysis method as described in (2). The binding of RBP and T4 to a mixture of tetramers 1-5 was indistinguishable from binding to wild type TTR. After separation of the five different tetramers as demonstrated in Fig. 1 (main paper), RP-HPLC analysis allows the subunit stoichiometry in each of the five TTR tetramers to be quantified. SDS-PAGE coupled with a denistometry analysis provides an alternative approach to quantify the subunit stoichiometry (Supplemental fig. 1).

Supplemental Figure 1. Analysis of TTR tetramer composition can also be performed by SDS PAGE demonstrating subunit purity and stoichiometry. The protein bands were visualized by Coomassie staining and quantified by densiometry.

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2. pH-dependent Solubility of V30M/FT₂-T119M Tetramers.

Supplemental Figure 2. The pH-dependent solubility of isolated TTR tetramers (3.6 M) of defined V30M/FT₂-T119M subunit stoichiometry was evaluated by mixing a solution of 7.2 M TTR tetramer in 10 mM phosphate buffer (100 mM KCl, 1 mM EDTA (pH 7.0)) with an equal volume of 200 mM citrate (pH 3-3.4), acetate (pH 3.6-5.8) or phosphate (pH 6.2-7.0) buffer (100 mM KCl, 1 mM EDTA) followed by incubation for 72h. The buffer utilized depends on the final pH desired for the solubility evaluation. After incubation, the samples were centrifuged at 12000 g for 10 min and 50 L of the supernatant was mixed with 50 L of Coomassie G-250 protein stain (Pierce) in a microtiter plate to record the TTR concentration (absorbance 595 nm).

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3. Stability data

Supplemental Table 1. Summary of stability data from GdmSCN unfolding of TTR hybrid tetramers.
Variant	c1/2 (M)	Dc1/2(M)	DGNUH2O(kcal(mol-1)	- mNU
(kcal(mol-1(M-1)	DDGNUH2O(kcal(mol-1)	DDGNUGdmSCN(kcal(mol-1)
V30M/FT2-T119M
Tetramer 1	0.97	0	4.2	4.4	0	0
Tetramer 2	1.03	0.06	5.2	5.1	-1.0	-0.2
Tetramer 3	1.12	0.15	5.2	4.7	-1.0	-0.7
Tetramer 4	1.14	0.17	5.2	4.5	-1.0	-0.8
Tetramer 5	1.33	0.36	7.4	5.6	-3.2	-1.8
V30M/FT2-WT
Tetramer 1	0.97	0	4.2	4.4	0	0
Tetramer 6	0.92	-0.05	5.8	6.3	-1.6	0.3
Tetramer 7	0.99	0.02	5.7	5.8	-1.5	-0.1
Tetramer 8	1.02	0.05	4.2	4.1	0	-0.3
Tetramer 9	1.07	0.10	4.8	4.5	-0.6	-0.5
Unfolding data were fitted to a single transition as described in (3). The difference in free energy between V30M (tetramer 1) and the other tetramers, DDGNUGdmSCN, was compared at a concentration of 1.15 M GdmSCN for V30M/FT2-T119M tetramers and at a concentration of 1.00 M GdmSCN for V30M/FT2-WT (the chosen concentrations represent the midpoint of denaturation between the most stable and least stable variant, reducing the error that may arise from extrapolation to water).

4. In vitro Co-refolding of V30M/FT₂-T119M at 37°C.

Supplemental Figure 3. Equimolar concentrations of V30M and FT₂-T119M (3.6 M) were unfolded by dialysis against pH 2.0 solution (10 mM HCl, in dH₂O) at 4°C for 48 h. Refolding was initiated by mixing the unfolded proteins with equal volume of 100 mM phosphate buffer, pH 7.2 (resulting in a final pH of 7.0) containing 100 mM KCl, 1 mM EDTA and DTT. (A) The reconstitution was allowed to proceed for 54 h followed by analytical ion exchange chromatography (4). The resulting chromatogram showed an almost statistical distribution (1:4:6:4:1) of tetramers. (B) For comparison, the chromatogram of V30M and FT₂-T119M incubated under native conditions for 168 h at 37°C is shown. This sample exhibits very little subunit exchange, under conditions simulating physiological.

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5. Experimental details.

A. Expression and purification of hybrid TTR tetramers

Expression of hybrid TTR was induced using 1 mM IPTG at an OD of 0.8 to 1.2 at 660 nm, with TTR purification accomplished as described previously (5) with an additional gel filtration step (superdex 75). Isolation of the hybrid tetramers was accomplished by preparative anion exchange chromatography using a source 15Q column (40 ml) employing a 20 to 43% gradient of NaCl in 25 mM Tris HCl (pH 8.0) containing 1 mM EDTA.

B. Fibril formation experiments

The following fibril formation assay is based on previously published protocols (5). Amyloid fibril samples were prepared by mixing a solution of 7.2 M TTR tetramer in 10 mM phosphate buffer (100 mM KCl, 1 mM EDTA (pH 7.0)) with an equal volume of 200 mM Na-Acetate buffer (100 mM KCl 1 mM EDTA (pH 4.3)), resulting in a TTR solution (3.6 M) at pH 4.4. These samples were incubated for 72 h at 37°C and the turbidity (400 nm) of the suspension was measured. The samples were thereafter centrifuged at 12,000g for 10 min and the pellet was resuspended (800 l of 50 mM phosphate buffer pH 7.0 containing 100 mM KCl, 1 mM EDTA, 1 mM DTT) and 4 L of a 2 mM thioflavin T (ThT) solution was added. The emission intensity of ThT at 482 nm, characteristic of ThT bound to amyloid fibrils (6), was recorded (excitation 440 nm). The TTR deposits made by the acidification protocol outlined above, were compared to TTR fibrillization orchestrated by organic solvent treatment (the most efficient method known (7)). Electron microscopy and thioflavin T binding revealed that pH 4.4 incubation produced deposits that display 50 % fibrillar characteristics relative to organic solvent induced fibrillization, the remainder being amorphous aggregates.

C. T119M monomers for inclusion into V30M rich tetramers.

T119M (0.30 mg/mL) (or FT₂-T119M) was unfolded in 2 mL of 6.5 M GdmCl at 4°C for 48 h. Refolding was initiated by rapid dilution (32.5-fold) to 0.2 M GdmCl, yielding a T119M concentration of 0.01 mg/mL. This procedure allows refolding of the T119M subunits but prevents their self-assembly into tetramers (unpublished results). The T119M monomer was concentrated to a volume of 11 mL (remains mostly monomeric) and an equimolar amount of tetramer 1 (0.60 mg) was added at 4°C. The mixture was dialyzed for 24 h at 4°C versus 10 mM phosphate buffer (pH 7.0) containing 100 mM KCl, 1 mM EDTA and 1 mM DTT. Quantification of subunit exchange was accomplished by analytical ion exchange chromatography as described in (4). The samples were concentrated to a final V30M protein concentration of 0.4 mg/mL. Samples (450 l) were thereafter mixed with an equal volume of fibril assay buffer composed of 200 mM Na-acetate, pH 4.4, 100 mM KCl and 1 mM EDTA. The TTR samples with differing extents of subunit exchange were subjected to amyloidogenic conditions and quantified as described herein. All the in vitro exchanged samples were compared to controls containing the same amount of non-exchanged V30M and FT₂-T119M or T119M tetramers. No effect on the amyloidogenicity of tetramer 1 was detected by merely adding equimolar tetramer 5 or T119M homotetramer.

References

1. J. T. White, K. P. Chiang, J. W. Kelly, unpublished.
2. H. E. Purkey, M. I. Dorrell, J. W. Kelly, Proc. Natl. Acad Sci. U.S.A. 98, 5566 (2001).
3. M. M. Santoro, D. W. Bolen, Biochemistry 27, 8063 (1988).
4. F. Schneider, P. Hammarström, J. W. Kelly, Protein Science 10, 1606 (2001).
5. Z. Lai, W. Colon, J. W. Kelly, Biochemistry 35, 6470 (1996).
6. H. Naiki, K. Higuchi, M. Hosokawa, T. Takeda, Anal. Biochem. 177, 244 (1989).
7. P. Hammarström, J. W. Kelly, unpublished.