Kinetic control in protein folding for light chain amyloidosis and the differential effects of somatic mutations

Abstract

Light chain amyloidosis is a devastating disease where immunoglobulin light chains form amyloid fibrils, resulting in organ dysfunction and death. Previous studies have shown a direct correlation between the protein thermodynamic stability and the propensity for amyloid formation for some proteins involved in light chain amyloidosis. Here we investigate the effect of somatic mutations on protein stability and in vitro fibril formation of single and double restorative mutants of the protein AL-103 compared to the wild-type germline control protein. A scan rate dependence and hysteresis in the thermal unfolding and refolding was observed for all proteins. This indicates that the unfolding/refolding reaction is kinetically determined with different kinetic constants for unfolding and refolding even though the process remains experimentally reversible. Our structural analysis of AL-103 and AL-103 delP95aIns suggests a kinetic coupling of the unfolding/refolding process with cis-trans prolyl isomerization. Our data reveal that the deletion of proline 95a (AL-103 delP95aIns), which removes the trans-cis di-proline motif present in the patient protein AL-103, results in a dramatic increment in the thermodynamic stability and a significant delay in fibril formation kinetics with respect to AL-103. Fibril formation is pH dependent; all proteins form fibrils at pH2; reactions become slower and more stochastic as the pH increases up to pH7. Based on these results, we propose that, in addition to thermodynamic stability, kinetic stability (possibly influenced by the presence of cis proline 95a) plays a major role in the AL-103 amyloid fibril formation process.

Keywords: CDR; ThT; amyloid fibril formation; complementarity-determining region; kinetic two-state model; light chain amyloidosis; protein thermodynamic and kinetic stability; scan rate dependence and hysteresis; thioflavin T.

Fig. 1. Structure, sequence, and spectroscopic properties of AL-103

(a) Structural alignment of x-ray crystal structures of κI O18/O8 germline (gold), AL-103 (red) and AL-103 del95aIns (blue) (PDB codes: 2Q20, 3DVI and 4K07 respectively). Highly conserved single Tryptophan residue at position 35 (black sticks) and disulfide bridge (gray sticks) are shown. (b) Structure-based multiple sequence alignment of κI O18/O8 and AL-103. AL-103 somatic mutations are highlighted. Three of the four AL-103 mutations are clustered in the CDR3 and β-strand G, while the fourth (N34I) is in the dimer interface on β-strand C. (c) Fluorescence (native, filled circles; unfolded, open circles) and (d) Far-UV CD spectra of AL-103.

Fig. 2. Hysteresis in thermal unfolding/refolding transitions as a function of scan rate

Thermal unfolding (filled circles) and refolding (open triangles) transitions were obtained at different scan rates (0.5°C/min, black; 2.5°C/min; olive green) of (a) AL-103 H92D, (b) AL-103, (c) AL-103 P100Q, (d) AL-103 H92D-I34N, (e) AL-103 I34N, (f) κI O18/O8 and (g) AL-103 del95aIns following the change in molar ellipticity at 217nm.

Fig. 3. Scan rate dependence of thermal unfolding/refolding

(a) AL-103 H92D, (b) AL-103, (c) AL-103 P100Q, (d) AL-103 H92D-I34N, (e) AL-103 I34N, (f) κI O18/O8 and (g) AL-103 del95aIns. Apparent Tm values calculated from unfolding experiments are shown in red, those corresponding to refolding experiments are shown in black. ΔTm was calculated as the difference between the apparent Tm from the unfolding transition- apparent Tm from the refolding transition from the fastest scan rate values for each protein. Tm values obtained from kinetic model analysis (filled star) are in agreement with values obtained from model-free analysis (open star) within the error. Continuous lines represent the best fit to a straight line (n=30).

Fig. 4. Contribution of restorative mutants to the kinetic stability

(a) Correlation between Tm estimate from the model-free analysis and the Tm values obtained from kinetic two-state model. Continuous lines represent the best fitting. The 95% confidence interval is shown in black thin lines. (b) T* and (c) ΔH^‡ values for each protein characterized in this study. Labels in the bars show the average value (n=30). Values derived from unfolding experiments are shown in red, values from refolding experiments are shown in black.

Fig. 5. Thermal and urea-induced unfolding transitions

(a) Thermal unfolding curves following molar ellipticity change at 217 nm. (b) Urea unfolding curves at 4°C following change in fluorescence intensity at 350nm. Experimental data from AL-103 (red) and AL-103 restorative mutants (AL-103 H92D, black; AL-103 H92D-I34N, olive green; AL-103 P100Q, orange; AL-103 I34N, blue; AL-103 del95aIns, green) (n=3). Continuous lines represent the best fit to a two-state model; see Table 1 for thermodynamic parameters. Data from κI O18/O8 (dark red) was calculated using thermodynamic parameters from Baden et al. 2008.

Fig. 6. In vitro fibril formation assay

(a) Average fibril formation kinetic traces of κI O18/O8 (dark red), AL-103 (red) and AL-103 restorative mutants (AL-103 H92D, black; AL-103 H92D-I34N, green olive; AL-103 P100Q, orange; AL-103 I34N, blue; AL-103 del95aIns, green) at pH 2. Average t₅₀ values (mean ± SE) were obtained by fitting each triplicate to Boltzmann equation. Continuous lines represent the best fitting. (b) pH dependence of fibril formation kinetics. Absence of error bar indicates that only one triplicate reaction increased to three-fold ThT fluorescence (>200,000 A.U). *p-value<0.005 with respect to AL-103.

Fig. 7. Electron microscopy images of fibrils formed at pH 2

(a) AL-103 H92D. (b) AL-103. (c) AL-103 P100Q. (d) AL-103 H92D-I34N. (e) AL-103 I34N. (f) κI O18/O8 and (g) AL-103 del95aIns. Scale bars represent 200 nm.

Fig. 8. Correlation of fibril formation vs. protein stability of AL-103 restorative mutants

(a) T* value vs t₅₀ value (hours). (b) ΔG^‡_unf (kcal/mol) at 37°C vs rates of fibril formation or t₅₀ value (hours). t₅₀ value where obtained from fibril formation kinetics at pH 2, 37°C.

Fig. 9. Electron density for AL-103 del P95aIns CDR3 region

Electron density map (2Fo-Fc at 1σ contouring) for the Proline 95 region of AL-103 del P95aIns. P95 is labeled.