Determination of methyl C 13 - N 15 dipolar couplings in peptidesand proteins by three-dimensional and four-dimensional magic-angle spinning solid-state NMR spectroscopy

We describe three- and four-dimensional semiconstant-time transferred echo double resonance (SCT-TEDOR) magic-angle spinning solid-state nuclear magnetic resonance (NMR) experiments for the simultaneous measurement of multiple long-range N 15 - C methyl 13 dipolar couplings in uniformly C 13 , N 15 -enriched peptides and proteins with high resolution and sensitivity. The methods take advantage of C 13 spin topologies characteristic of the side-chain methyl groups in amino acids alanine, isoleucine, leucine, methionine, threonine, and valine to encode up to three distinct frequencies ( N 15 - C methyl 13 dipolar coupling, N 15 chemical shift, and C methyl 13 chemical shift) within a single SCT evolution period of initial duration ∼ 1 ∕ J C C 1 (where J C C 1 ≈ 35 Hz , is the one-bond C methyl 13 - C 13 J -coupling) while concurrently suppressing the modulation of NMR coherences due to C 13 - C 13 and N 15 - C 13 J -couplings and transverse relaxation. The SCT-TEDOR schemes offer several important advantages over previous methods of this type. First, significant (approximately twofold to threefold) gains in experimental sensitivity can be realized for weak N 15 - C methyl 13 dipolar couplings (corresponding to structurally interesting, ∼ 3.5 Å or longer, distances) and typical C methyl 13 transverse relaxation rates. Second, the entire SCT evolution period can be used for C methyl 13 and/or N 15 frequency encoding, leading to increased spectral resolution with minimal additional coherence decay. Third, the experiments are inherently "methyl selective," which results in simplified NMR spectra and obviates the use of frequency-selective pulses or other spectral filtering techniques. Finally, the N 15 - C 13 cross-peak buildup trajectories are purely dipolar in nature (i.e., not influenced by J -couplings or relaxation), which enables the straightforward extraction of N 15 - C methyl 13 distances using an analytical model. The SCT-TEDOR experiments are demonstrated on a uniformly C 13 , N 15 -labeled peptide, N-acetyl-valine, and a 56 amino acid protein, B1 immunoglobulin-binding domain of protein G (GB1), where the measured N 15 - C methyl 13 dipolar couplings provide site-specific information about side-chain dihedral angles and the packing of protein molecules in the crystal lattice.