Collagen Structure-Function Relationships from Solid-State NMR Spectroscopy.

The extracellular matrix of a tissue is as important to life as the cells within it. Its detailed molecular structure defines the environment of a tissue's cells and thus their properties, including differentiation and metabolic status. Collagen proteins are the major component of extracellular matrices. Self-assembled collagen fibrils provide both specific mechanical properties to handle external stresses on tissues and, at the molecular level, well-defined protein binding sites to interact with cells. How the cell-matrix interactions are maintained against the stresses on the tissue is an important and as yet unanswered question. Similarly, how collagen molecular and fibrillar structures change in aging and disease is a crucial open question. Solid-state NMR spectroscopy offers insight into collagen molecular conformation in intact in vivo and in vitro tissues, and in this Account we review how NMR spectroscopy is beginning to provide answers to these questions. In vivo 13C,15N labeling of the extracellular matrix has given insight into collagen molecular dynamics and generated multidimensional NMR "fingerprints" of collagen molecular structure that allow comparison of local collagen conformation between tissues. NMR studies have shown that charged collagen residues (Lys, Arg) adopt extended-side-chain conformations in the fibrillar structure to facilitate charge-charge interactions between neighboring collagen molecules, while hydrophobic residues (Leu, Ile) fold along the collagen molecular axis to minimize the hydrophobic area exposed to surrounding water. Detailed NMR and molecular modeling work has shown that the abundant Gly-Pro-Hyp (Hyp = hydroxyproline) triplets in collagen triple helices confer well-defined flexibility because the proline is conformationally metastable, in contrast to the expectation that these triplets confer structural rigidity. The alignment of the Gly-Pro-Hyp triplets within the fibril structure means that the Gly-Pro-Hyp molecular flexibility generates fibril flexibility. The fibrillar bands of Gly-Pro-Hyp are highly correlated with collagen ligand binding sites, leading to the hypothesis that the fibril alignment of Gly-Pro-Hyp triplets is essential to protect collagen-ligand binding against external stresses on the tissue. Non-enzymatic chemistry between collagen side-chain amine groups (Lys, Arg) and reducing sugars-glycation-is an important source of matrix structural change in aging and disease. Glycation leads to stiffening of collagen fibrils, which is widely speculated to be the result of intermolecular cross-linking. The chemistry of non-enzymatic glycation has been extensively detailed through NMR studies and has been shown to lead to side-chain modifications as the majority reaction products, rather than intermolecular cross-links, with resultant molecular misalignment in the fibrils. Thus, a picture is beginning to emerge in which collagen glycation causes stiffening through misalignment of collagen molecular flexible regions rather than intermolecular cross-linking, meaning that new thinking is needed on how to alleviate collagen structural changes in aging and disease.