Determination of Multiple Torsion-Angle Constraints in U−13C,15N-Labeled Peptides: 3D 1H−15N−13C−1H Dipolar Chemical Shift NMR Spectroscopy in Rotating Solids

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Abstract
We demonstrate constraint of peptide backbone and side-chain conformation with 3D 1H−15N−13C−1H dipolar chemical shift, magic-angle spinning NMR experiments. In these experiments, polarization is transferred from 15N[i] by ramped SPECIFIC cross polarization to the 13Cα[i], 13Cβ[i], and 13Cα[i − 1] resonances and evolves coherently under the correlated 1H−15N and 1H−13C dipolar couplings. The resulting set of frequency-labeled 15N1H−13C1H dipolar spectra depend strongly upon the molecular torsion angles φ[i], χ1[i], and ψ[i − 1]. To interpret the data with high precision, we considered the effects of weakly coupled protons and differential relaxation of proton coherences via an average Liouvillian theory formalism for multispin clusters and employed average Hamiltonian theory to describe the transfer of 15N polarization to three coupled 13C spins (13Cα[i], 13Cβ[i], and 13Cα[i − 1]). Degeneracies in the conformational solution space were minimized by combining data from multiple 15N1H−13C1H line shapes and analogous data from other 3D 1H−13Cα−13Cβ−1H (χ1), 15N−13Cα−13C‘−15N (ψ), and 1H−15N[i]−15N[i + 1]−1H (φ, ψ) experiments. The method is demonstrated here with studies of the uniformly 13C,15N-labeled solid tripeptide N-formyl-Met-Leu-Phe-OH, where the combined data constrains a total of eight torsion angles (three φ, three χ1, and two ψ): φ(Met) = −146°, ψ(Met) = 159°, χ1(Met) = −85°, φ(Leu) = −90°, ψ(Leu) = −40°, χ1(Leu) = −59°, φ(Phe) = −166°, and χ1(Phe) = 56°. The high sensitivity and dynamic range of the 3D experiments and the data analysis methods provided here will permit immediate application to larger peptides and proteins when sufficient resolution is available in the 15N−13C chemical shift correlation spectra.

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