Probing the kinetic stabilities of Friedreich's ataxia clinical variants using a solid phase GroEL chaperonin capture platform

Abstract

Numerous human diseases are caused by protein folding defects where the protein may become more susceptible to degradation or aggregation. Aberrant protein folding can affect the kinetic stability of the proteins even if these proteins appear to be soluble in vivo. Experimental discrimination between functional properly folded and misfolded nonfunctional conformers is not always straightforward at near physiological conditions. The differences in the kinetic behavior of two initially folded frataxin clinical variants were examined using a high affinity chaperonin kinetic trap approach at 25 °C. The kinetically stable wild type frataxin (FXN) shows no visible partitioning onto the chaperonin. In contrast, the clinical variants FXN-p.Asp122Tyr and FXN-p.Ile154Phe kinetically populate partial folded forms that tightly bind the GroEL chaperonin platform. The initially soluble FXN-p.Ile154Phe variant partitions onto GroEL more rapidly and is more kinetically liable. These differences in kinetic stability were confirmed using differential scanning fluorimetry. The kinetic and aggregation stability differences of these variants may lead to the distinct functional impairments described in Friedreich's ataxia, the neurodegenerative disease associated to frataxin functional deficiency. This chaperonin platform approach may be useful for identifying small molecule stabilizers since stabilizing ligands to frataxin variants should lead to a concomitant decrease in chaperonin binding.

Figure 1

Comparison between the stability profile of wild type frataxin (FXN) and two clinical mutants FXN-p.Asp122Tyr and FXN-p.Ile154Phe. (A) Thermal denaturation curves of (■) FXN (Tm = 66.3 ± 0.1 °C), (○) FXN-p.Asp122Tyr (Tm = 50.4 ± 0.1 °C) and (Δ) FXN-p.Ile154Phe (Tm = 50.7 ± 0.1 °C) (Curves redrawn from [11] to highlight variants used herein) demonstrate differences between wt FXN and the two mutants; (B–D) Effect of temperature on Frataxin partitioning profiles (starting concentration 2 µM Frataxin) onto 2 µM immobilized GroEL oligomer beads in the presence of 1 M Urea. The partitioning of (B) Wild type FXN, (C) FXN-p.Asp122Tyr (circles,) and (D) FXN-p.Ile154Phe (triangles) was monitored by UV-visible spectroscopy at 25 °C, 37 °C and 45 °C to demonstrate that stability differences observed in (A) can be recapitulated with the GroEL chaperonin sink assay.

Figure 2

Effect of GroEL concentration on frataxin clinical variants partitioning profiles at 45 °C, 1 M urea. Frataxin Partitioning (2 µM) was analyzed using different estimated GroEL oligomer concentrations: control-no GroEL (●), 0.5 µM (■), 1 µM (▲), 2 µM (▼) and 3 µM (♦); (A) FXN-p.Ile154Phe (FXN I154F) and (B) FXN-p.Asp122Tyr (FXN D122Y). Each time point consisted of three separate spectroscopic measurements and the error bars represent ± 1 S.D. The pseudo first order fits to the data are represented by the red dotted lines. The values for each fit are listed in Supplementary Table S1; (C) The individual time point data representing the remaining frataxin in solution for each GroEL concentration series were fit to a pseudo first order kinetic profile. The pseudo first order partitioning rate increases as the GroEL concentration increases. The GroEL concentration is in excess of the amount of the partially folded frataxin concentration (not the total frataxin concentration). The remaining FXN concentrations declining with time, i.e., the partitioning curves (in A and B) were fit to a pseudo-first order relationship to obtain the partitioning rates. A plot of the macroscopic partitioning pseudo first order rate vs. estimated GroEL concentration follows a hyperbolic relationship that tends towards saturation at higher GroEL concentrations.

Figure 3

In the absence of GroEL beads, general FXN-p.Ile154Phe aggregation (2 µM) occurs in solution alone (no osmolytes) (A) in the presence of 4M Glycerol (B) or 1 M TMAO (C). FXN-p.Ile154Phe was incubated at 45 °C for 60 min in the absence and presence of the different osmolytes and the UV absorbance spectra at time 0 min and 60 min are represented. The spectra in the presence of 1 M TMAO shows a larger increase in light scattering contributions as assessed by the increase in the general baseline from general protein aggregation compared without osmolytes (A) or with glycerol (B). The 1 and 60 min spectra of FXN-p.Asp122Tyr under all three above conditions do not show a significant wavelength dependent shift in the baseline due to light scattering from aggregation (Supplementary Figure S3).

Figure 4

Differential Scanning fluorimetry (DSF) of native and variant frataxins in the absence and presence of TriMethylAmine N-oxide (TMAO). The concentration of frataxin was 4 µM. The dye Sypro orange was used to probe the temperature dependent protein unfolding reaction. The starting apparent kinetic T_m values without osmolyte were 55.1 °C for wild type FXN (A), 48.8 °C for FXN-p.Asp122Tyr (B) and 39.1 °C for FXN-p.Ile154Phe (C) (see also Table 1). Panels (A–C) Melting curves for the three variants, measured by Differential Scanning Fluorimetry in the presence of increasing concentrations of two osmolytes. Panel (D). TMAO efficiently increases the T_m values of all FXN variants in a concentration dependent way. (■) FXN wild type, (○) FXN-p.Asp122Tyr and (∆) FXN-p.Ile154Phe.

Figure 5

Effect of chemical chaperones on early folding events. (A) SDS/PAGE gels obtained from E. coli lysates expressing frataxin. For each protein variant, the soluble (S) and insoluble (P) fractions are shown; (B) Semi-quantitative analysis of the relative proportion of frataxin present in the soluble and insoluble fractions, obtained from densitometric analysis of gel bands (n = 3), allowed the determination of the protein expressed in the soluble form; (C) Variation of the folding efficiency induced by the presence of chemical chaperones (increasing concentrations of TMAO and glycerol). After the densitometric analysis of the gel bands, the ratio between the amount of soluble protein present in the presence and absence of the compounds were determined. The Log2 of this ratio is here shown to highlight the variation observed.

Figure 6

Partitioning profile changes of FXN-p.Ile154Phe (FXN-I154F) and FXN-p.Asp122Tyr (FXN-D122Y) (both at 2 µM) in the absence (solid triangle) and presence (solid box) of 2 µM GroEL beads in the presence of 4 M glycerol ((A,C), respectively) or 1 M TMAO ((B,D), respectively). In both instances, the non-denaturing concentration of urea is 1 M at 45 °C. (A,C). The osmolyte solubility/partitioning profiles were compared with time dependent solubility changes without osmolytes.