2D and 3D ¹⁵N-¹³C-¹³C NMR chemical shift correlation spectroscopy of solids: Assignment of MAS spectra of peptides

Strategies are discussed for resolving and assigning peptide backbone and side chain resonances in uniformly ¹³C, ¹⁵N-labeled solid peptides. Methods for 2D ¹³C-¹³C, ¹⁵N-(¹³C)-¹³C, and 3D ¹⁵N-¹³C-¹³C chemical shift correlation spectroscopy are demonstrated in the chemotactic tripeptide N-formyl-[U-¹³C, ¹⁵N]-Met-Leu-Phe-OH (MLF). Band-selective heteronuclear double-cross polarization (DCP) and γ-encoded homonuclear double-quantum mixing provide large improvements in sensitivity relative to previously published methods. Directional transfers from amide ¹⁵N to ¹³C' or ¹³C(α) resonances provide two- to 3-fold improvements in signal intensity on the observed ¹³C spin, in comparison to broadband DCP. Similarly, homonuclear ¹³C¹³C transfer is enhanced by use of the rotating frame sequence SPC-5; backbone-to-side chain polarization transfers are achieved with especially high efficiency. Furthermore, the double-quantum nature of the homonuclear transfer permits straightforward classification of C', C(α), C(β), and C(γ) signals, on the basis of the sign of the cross-peaks. The experiments described here include optimized solid-state analogues of the solution-state schemes commonly employed for amino acid identification and sequential backbone assignment. We expect that these experiments will facilitate application of 2D and 3D correlation methods to assignment of larger solid peptides and proteins.