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.
ASJC Scopus subject areas
- Colloid and Surface Chemistry