Biomolecular structures elucidated by cOLD magic angle spinning NMR with dynamic nuclear polarization

Project Information

BOLD-NMR

Grant agreement ID: 795423

DOI

10.3030/795423

Project terminated on 10 October 2019

EC signature date 26 March 2018

Start date 26 April 2018

End date 25 April 2020

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 173 076,00

EU contribution

€ 173 076,00

Coordinated by

COMMISSARIAT A L ENERGIE ATOMIQUE ET AUX ENERGIES ALTERNATIVES
France

Periodic Reporting for period 1 - BOLD-NMR (Biomolecular structures elucidated by cOLD magic angle spinning NMR with dynamic nuclear polarization)

Reporting period: 2018-04-26 to 2020-04-25

Summary of the context and overall objectives of the project

Protein aggregates are the hallmark for many incurable protein-misfolding disorders and continue to be challenging targets for structural studies. Magic angle spinning solid-state NMR (MAS ssNMR) is particularly adept at characterizing these types of self-assembled samples. Given the correlations between atomic structure of aggregate polymorphs and their toxicity, and the influences of the cellular milieu on aggregate formation, it is imperative to examine protein aggregates under native conditions. However, the reliance of MAS ssNMR on multidimensional 13C/15N correlation spectroscopy has limited or prevented applications to protein aggregates that are hard or impossible to isotopically label, such as animal- or patient-derived samples. The global objective of this project was to develop a new approach for the structural characterization of biomolecules, by MAS ssNMR, which will be compatible with samples derived from native sources, as for example fibrillar plaque obtained from tissue. This would be invaluable in understanding the mechanisms of fibril formation in neurodegenerative diseases. The approach for this project relied on the use of samples at natural isotopic abundance (NA), studied with an emerging technique ULT-MAS-DNP (Ultra-Low Temperature Magic Angle Spinning Dynamic Nuclear Polarization). Moreover, working at NA has facilitated the measurement of inter-molecular distances, a necessity for high-resolution structural characterization. This ULT-MAS-DNP methodology was applied and further developed by investigating polyglutamine (polyQ) fibrils implicated that are Huntington’s disease.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

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.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The results from this project demonstrated that it is feasible to obtain multidimensional 13C/15N spectra and precise structural restraints of protein aggregates at NA, which was beyond the state-of-the-art for MAS ssNMR experiments and characterization techniques in general. These experiments were extended to studies of samples under fast-MAS in 1.3 mm diameter rotors. This is a milestone in achieving the long-term goal (structural characterization of ex-vivo-derived samples) as the amount of sample that can be expected from these sources is likely in the sub-milligram range. In addition, the precise distance restraints obtained with this methodology lends to the idea that polyQ fibrils form an anti-parallel beta sheet configuration in the amyloid core. To date, there is no high-resolution structure of polyglutamine fibrils and, as such, the latter result is an important descriptor in developing a model of these protein aggregates. The work completed will provide a basis for the scientific community to study protein aggregates in native conditions. This will enable a more informed description of structural properties that can lead to neurodegenerative diseases. The potential societal implications are vast. Economically, progress in ULT-MAS-DNP and DNP with fast-MAS will benefit not only NMR industry – which will trickle-down to the academic NMR community – but also various other industries where structural characterization is crucial to progress.

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