Objective (1) largely focused on the development and application of spectroscopic methods for the structural characterization of polyQ fibrils at NA by solid-state NMR and enabled by DNP. The inherently low sensitivity of NMR coupled with the low natural abundance of the NMR active nuclei 13C and 15N (1.1 % and 0.4 %, respectively) necessitated the use of MAS-DNP. In objective 1 two types of samples were primarily studied: polyQ fibrils composed of the model peptide D2Q15K2 and the disease-relevant exon 1 mutant huntingtin protein with a 44 glutamine expansion (Htt-Q44). The large enhancements provided by MAS-DNP enabled the acquisition of multidimensional 13C – 13C and 13C -15N correlation spectra at NA. The latter experiments resulted in 13C and 15N chemical shift assignment of the polyQ amyloid core and oligo-proline regions of D2Q15K2 and Htt-Q44 fibrils. The assignments obtained verified that the amyloid core is composed primarily of a beta sheet conformation and that the oligoproline regions either adopted a type-II polyproline helix or remained unstructured. In addition, working at NA enabled the measurement of long-range distance restraints (i.e. 3-8 Å), via dipolar recoupling experiments. Because most 13C spin pairs are isolated at NA, this circumvents the issue of dipolar truncation. This methodology was further developed and applied to polyQ fibrils where it was notably shown that an antiparallel beta-strand configuration best described the measured restraints by dipolar recoupling experiments. These results represent significant progress in the study of polyQ fibril structure and polymorphism, as no high-resolution structure of these aggregates is available to date.
Objective (2) was to implement a 1.3/0.7 mm ULT-MAS-DNP probe. In particular, it was focused on making a closed-loop helium cryostat in combination with a fast ULT-MAS-DNP probe operational at ULT (~ 30 – 80 K). The improved sensitivity of ULT-MAS-DNP instrumentation that operates in the 30 - 80 K regime would enable experiments that are even more demanding from a sensitivity standpoint. One example of such experiments is heteronuclear 13C – 15N distance measurements of polyQ fibrils at NA, where 13C - 15N spin pairs only occur with a probability of 0.004 %. To date, the closed-loop helium cryostat, was tested with a commercial 3.2 mm MAS-DNP probe. The tests successfully demonstrated MAS of a 3.2 mm rotor up to 20 kHz and acquisition of DNP-enhanced spectra at 9 kHz MAS and 80 K on a small molecule suspension. To prevent helium leaks and ensure the feasibility of continuous operation of a closed-loop system, a helium-tight MAS-DNP probe has been constructed and is under final development within the lab. This has been done in collaboration with cryogenic engineers at CEA Grenoble and industrial partners. It is anticipated that initial MAS-DNP experiments utilizing the custom built probe will be conducted in the coming months.
Objective (3) centered on developing and characterizing polarizing agents that were efficient for DNP under fast-MAS and apply them for further characterization of polyQ fibrils at NA. In this aim, we investigated how efficient the polarizing agents AMUPol and AsymPolPOK were when used to enhance NA polyQ fibrils at 40 kHz MAS. AsymPolPOK resulted in larger sensitivity gains than AMUPol. This was due to the fast buildup time of hyperpolarization and low depolarization induced by AsymPolPOK. The large gains in sensitivity yielded from using AsymPolPOK enabled us to acquire 13C-13C correlation spectra of only 1 mg of fibrils at NA. One key advantage to performing these 13C-13C correlation experiments under fast-MAS was the increase in achievable bandwidth of this particular experiment, which is proportional to MAS rate. Therefore, NA measurements under fast-MAS enabled us to obtain additional structural restraints on polyQ fibrils that were previously inaccessible, such as side-chain orientations. Working at NA under fast-MAS rates is particularly challenging because 1.3 mm rotor diameters are required to achieve 40 kHz MAS. As such, this drastically reduces the amount of sample that can be packed into a rotor, thereby decreasing the sensitivity of the experiment. However, as we demonstrated, sufficient sensitivity gains could be obtained by using MAS-DNP and judicious choice of polarizing agent.
