A30P and A53T mutations destabilize αS

Residual dipolar couplings are exquisitely sensitive to bond vector orientation and can be measured in a weakly alignedprotein, for which the large internuclear dipolar interactions no longer average to zero (Bax and Grzesiek, 1993). As we reported in Chapter 4, RDCs identified five different domains in wt αS with different degrees of residual structure, in some cases long-range-coded (domain I: residues 1-28; domain II: residues 33-65; domain III:

residues 70-88; domain IV: residues 95-105; and domain V: the C-terminus). In particular, RDCs probed a hydrophobic core comprising the C-terminus and domain III and IV, which resulted to be a native gatekeeper for avoiding αS aggregation. We intended to measure this NMR-derived parameter in order to obtain further evidence for differences between the backbone conformations populated by both mutant proteins.

RDCs in PD-linked mutant αS were first observed by dissolving A30P and A53T αS mutants in a dilute liquid crystalline phase of 5% C8E5/octanol. No significant differences in the chemical shifts of spectra in C8E5/octanol and free in solution were observed, suggesting that the anisotropic phase did not appreciably perturb the ensemble of αS conformations. In general reduced couplings were observed for many residues in both genetic mutants when compared with wt αS (Figure 5.7). Since both variant are just single point mutations, and due to the conservation of solution and conditions of anisotropic phase preparation, the differences are not ascribed to variations in protein alignment, rather are likely caused by changes at the level of the conformations sampled by these proteins with respect to the wt.

Figure 5.7. RDCs in αS genetic mutants. Region of the ¹H¹⁵N- in phase-anti phase (IPAP)-HSQC spectrum corresponding to αS wt (left) and αS A30P (right) aligned in 5% C8E5/octanol mixture, showing the ¹DNH couplings for Glu⁸³ (E83) and

Asp¹¹⁹ (D119). The upfield doublet components (anti-phase) are indicated in red, while the downfield components (in-phase) in blue.

RDCs in A30P and A53T mutant αS were predominantly positive with regions displaying relatively large RDCs separated by residues with RDCs close to zero (Figure 5.8A, 5.8B), and slightly negative couplings were observed for some residues.

Figure 5.8. Residual dipolar couplings in mutant αS. Backbone N-H dipolar couplings, ¹DNH, were measured in A30P and A53T mutant αS oriented in a 5%

C8E5/octanol mixture at 15 °C. Non-zero dipolar couplings are indicative of structural

and motional restrictions of the protein backbone. A. DNH profile for A30P αS (blue) and wt αS (black) in buffer A. B. DNH profile for A53T αS (green) and wt αS (black) in buffer A. C. DNH couplings in the C-terminus of A30P αS in buffer A (red) and of wt αS in buffer A + 8 M urea (dark blue). D. DNH couplings in the C-terminus of A53T αS in buffer A (red) and of wt αS in buffer A + 4 M urea (blue). RDC domains identified in wt αS (domain I: residues 1-28; domain II: residues 33-65; domain III: residues 70-88; domain IV: residues 95-105; and domain V: the C-terminus) (Chapter 4) are indicated.

Despite the reduced values, the profiles corresponding to the mutant dipolar couplings correlate well with the RDC domain pattern of the wt protein. However, some differences were observed in particular domains. In contrast to wt αS, the RDCs for domain II (residues 50-62) and domain III (residues 70-94) were strongly reduced. In the C-terminal domain, the magnitudes of RDCs were comparable to those in the rest of the protein, as opposed to the RDC profile of wt αS, in which the values were almost twice as high. Rather the dipolar couplings of A30P αS were similar to those of wt αS in the presence of 8 M urea (Figure 5.8C), whereas the magnitude of RDCs of A53T αS was comparable to that of wt αS in the presence of 4 M urea (Figure 5.8D). Furthermore the profile of urea denatured A30P mutant is similar to wt αS, but still differs from the pattern observed in native solution conditions (Figure 5.9A).

In the vicinity of both missense mutations, the dipolar couplings were considerably reduced, suggesting an increased mobility in these regions. For the A30P mutation, the increased flexibility is in agreement with the reduced heteronuclear ¹⁵N R1ρ relaxation rates of backbone amide groups and a reduced helical propensity for domain I (residues 18-31) (Bussell and Eliezer, 2001).

In line with the alteration of long-range interactions by PD-linked mutations, as suggested previously by PRE measurements, RDCs also evidenced a very strong reduction of couplings in the hydrophobic NAC region and at the C-terminal domain of mutant αS. These results point toward a reduced shielding of the hydrophobic NAC region by the C-terminus in the A30P and A53T mutant αS.

As an important control of those experiments, the couplings for A18C αS, a non-amyloidogenic mutant of αS with an unaltered time course of aggregation (Chapter 1), were nearly identical to that of wt αS (Figure 5.9B).

Figure 5.9. Control data for residual dipolar couplings in PD-linked mutant αS. Backbone N-H dipolar couplings, DNH, were measured in αS variants oriented in a 5% C8E5/octanol mixture at 15 °C. A. DNHprofile of A30P αS (blue) and wt αS (black) both in buffer A + 8 M urea. B. DNHprofile of A18C αS (orange), a non-amyloidogenic mutant of αS with an unaltered time course of aggregation, and wt αS (black) both in buffer A.

In addition, the results presented in figure 5.8 were reproduced in Pf1 phages as a second alignment medium (Figure 5.10A, 5.10B), confirming in general our findings. There was, as well, an overall reduction of the RDCs values for the polypeptide chain, confirming that the populated conformers differ. However, the strong reduction of the couplings observed for the C-terminus in C8E5/octanol phase was shown in Pf1 media as a mild reduction, similarly to what we evidence upon raising the temperature on the wt protein, and confirming the reduced strength of long range interactions.

We further measured RDCs at higher temperature for the A30P mutant, in order to determine whether there was a difference between both proteins at physiological conditions.

When the temperature was raised to 37 °C we still spotted the reduced value of C-terminal dipolar couplings suggesting that the destabilizing effect of the genetic mutations is also significant at physiological conditions (Figure 5.10C).

Figure 5.10. Residual dipolar couplings in mutant αS oriented in Pf1 bacteriophage. A. DNH profile for A30P αS (blue) and wt αS (black). B. DNH profile for A53T αS (green) and wt αS (black). C. DNH profile for the residues corresponding to the C-terminus of A30P αS (blue and red) and wt αS (black) at 15 °C (left panel) and 37 °C (right panel).