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 13Cα resonances provide two- to 3-fold improvements in signal intensity on the observed 13C spin, in comparison to broadband DCP. Similarly, homonuclear 13C−13C 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‘, Cα, Cβ, and Cγ sig...