Cross-Polarization Schemes for Improved Heteronuclear Transfers Involving Labile Protons in Biomolecular Solution NMR

INEPT-based experiments are widely used for ¹H→¹⁵N transfers, but often fail when involving labile protons due to solvent exchanges. J-based cross polarization (CP) strategies offer a more efficient alternative to perform such transfers, particularly when leveraging the Hʷᵃᵗᵉʳ ↔ Hᴺ exchange process to boost the ¹H→¹⁵N transfer process. This leveraging, however, demands the simultaneous spin-locking of both Hʷᵃᵗᵉʳ and Hᴺ protons by a strong ¹H RF field, while fulfilling the γₕB₁,ₕ=γₙB₁,ₙ Hartmann-Hahn matching condition. Given the low value of γₙ/γₕ, however, these demands are often incompatible—particularly when experiments are executed by the power-limited cryogenic probes used in contemporary high field NMR. The present manuscript discusses CP alternatives that can alleviate this limitation, and evaluates their performance on urea, amino acids, and intrinsically disordered proteins. These alternatives include new CP variants based on frequency-swept and phase-modulated pulses, designed to simultaneously fulfill the aforementioned conflicting conditions. Their performances vis-à-vis current options are theoretically analyzed with Liouville-space simulations, and experimentally tested with double and triple resonance transfer experiments.ISSN:1433-7851ISSN:1521-3773ISSN:0570-083

1H, 13C and 15N chemical shift assignment of the stem-loops 5b + c from the 5′-UTR of SARS-CoV-2

The ongoing pandemic of the respiratory disease COVID-19 is caused by the SARS-CoV-2 (SCoV2) virus. SCoV2 is a member of the Betacoronavirus genus. The 30 kb positive sense, single stranded RNA genome of SCoV2 features 5'- and 3'-genomic ends that are highly conserved among Betacoronaviruses. These genomic ends contain structured cis-acting RNA elements, which are involved in the regulation of viral replication and translation. Structural information about these potential antiviral drug targets supports the development of novel classes of therapeutics against COVID-19. The highly conserved branched stem-loop 5 (SL5) found within the 5'-untranslated region (5'-UTR) consists of a basal stem and three stem-loops, namely SL5a, SL5b and SL5c. Both, SL5a and SL5b feature a 5'-UUUCGU-3' hexaloop that is also found among Alphacoronaviruses. Here, we report the extensive H-1, C-13 and N-15 resonance assignment of the 37 nucleotides (nts) long sequence spanning SL5b and SL5c (SL5b +c), as basis for further in-depth structural studies by solution NMR spectroscopy.ISSN:1874-270XISSN:1874-271