A novel model-free analysis of 13C NMR relaxation of alanine-methyl side-chain motions in peptides

Alanine residues in two peptide sequences derived from the βsheet domain of platelet factor-4: IA*TLK (PS) and TAQUA*TLK NGRKICLDLQA (P20), were synthesized with ₁₃C enriched in C and Cβ positions. 13C NMR relaxation measurements (proton coupled and decoupled) were performed at two NMR frequencies (150 and 62.5 MHz) and over a wide range of temperatures (5 to 75°C ) to study motional dynamics of the alanine side-chain methyl group and alanine side-chain-backbone motional correlations. Cross-correlation spectral densities, J_HCH(C), for C_βH₃ in both peptides are positive, indicating methyl-group rotational anisotropy. Various rotational models and model-free approaches have been used to analyze NMR relaxation data. The overall correlation times show a stronger temperature dependence in P20 than in PS, indicating the influence on alanine motions of folded conformational populations in P20. For analysis of alanine side-chain motions, an alternative model-free approach parameterized with a novel mixing parameter, A², that depends on the "geometry" of C_α-C_β and C_α-H bond rotations is proposed. By plotting the standard order parameter S² versus A², motional models may be visually differentiated. Molecular dynamics calculations were performed to compare C_α-C_β and C_α-H motions. Significant anticorrelated tp(t) and if/(t) backbone rotations can explain NMR relaxation data for P20.