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., (13)C, (15)N) and spin-1 (2)H, using phase-modulated deuterium dephasing pulses, provides a simple and efficient basis for obtaining peptide backbone torsion angles (phi, psi) 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 (13)C homonuclear interactions present during (13)C-observed, (2)H-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 (2)H(alpha)-alanine (in the tripeptide LAF) and the special case of (2)H(alpha)(2)-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 (13)C spin interactions, allowing determination of (phi, psi) backbone torsion angles.