Peptide torsion angle measurements: Effects of nondilute spin pairs on carbon-observed, deuterium-dephased PM5-REDOR

Reintroducing dipolar coupling between spin-1/2 nuclei (e.g., ¹³C, ¹⁵N) and spin-1 ²H, using phase-modulated deuterium dephasing pulses, provides a simple and efficient basis for obtaining peptide backbone torsion angles (φ, ψ) in specific stable-isotope enriched samples. Multiple homonuclear spin-1/2 interactions due to isotopic enrichment can arise between neighboring molecules or within a multiply labeled protein after folding. The consequences of ¹³C homonuclear interactions present during ¹³C-observed, ²H-dephased REDOR measurements are explored and the theoretical basis of the experimentally observed effects is investigated. Two tripeptides are taken to represent both the general case of ²H^α-alanine (in the tripeptide LAF) and the special case of ²H₂^α-glycine (in the tripeptide LGF). The lyophilized tripeptides exhibit narrowed spectral linewidths over time due to reduced conformational dispersion. This is due to a hydration process whereby a small fraction of peptides is reorienting and the bulk peptide fraction undergoes a conformational change. The new molecular packing arrangement lacks homonuclear ¹³C spin interactions, allowing determination of (φ, ψ) backbone torsion angles.