Assignment of aromatic side-chain spins and characterization of their distance restraints at fast MAS

Abstract

The assignment of aromatic side-chain spins has always been more challenging than assigning backbone and aliphatic spins. Selective labeling combined with mutagenesis has been the approach for assigning aromatic spins. This manuscript reports a method for assigning aromatic spins in a fully protonated protein by connecting them to the backbone atoms using a low-power TOBSY sequence. The pulse sequence employs residual polarization and sequential acquisitions techniques to record HN- and HC-detected spectra in a single experiment. The unambiguous assignment of aromatic spins also enables the characterization of 1H–1H distance restraints involving aromatic spins. Broadband (RFDR) and selective (BASS-SD) recoupling sequences were used to generate HN-ΗC, HC-HN and HC-HC restraints involving the side-chain proton spins of aromatic residues. This approach has been demonstrated on a fully protonated U-[13C,15N] labeled GB1 sample at 95–100 kHz MAS.

Keywords

• Aromatic side-chain assignments
• Multiple acquisitions
• 1H–1H distance restraints
• Fast MAS
• Solid-state NMR