Characterization of π-Interactions in Native Collagen by Solid-State NMR Spectroscopy

Collagen, a key extracellular matrix (ECM) protein of bone, provides connective tissues with strength and cohesion through its unique triple-helical structure, whose disruption is linked to numerous diseases and aging. The nanoscale organization of collagen within native bone ECM remains poorly understood. In this study, we employ high-resolution fast magic-angle spinning (MAS) solid-state NMR (ssNMR) spectroscopy to investigate collagen structure directly within the native bone matrix. Using two-dimensional (2D) ¹H-detected ¹³C-¹H double cross-polarization experiments at 70 kHz MAS, we detect signals from low-abundance residues and uncover previously unresolved inter-residue correlations in the aliphatic region. These proximities suggest potential π-interactions between aromatic residues and anionic or imino acids within the triple helix. Such interactions could provide additional stabilizing forces that are frequently overlooked in hydrogen bond-centered structural models. Our results reveal previously missing insights into the chemico-physical basis of collagen structural stabilization in the native ECM, laying the foundation for understanding disease-related structural changes and guiding the design of biomimetic materials to advance tissue engineering.