NMR Monitoring of Chain-Specific Stability in Heterotrimeric Collagen Peptides

摘要

Triple-helical peptides serve as valuable models for collagen, a critical protein in normal tissue structure and in disease. Close packing of three polyproline-like chains in the collagen triple-helix structure generates the requirement for glycine as every third residue, (Gly-X-Y)_n. Peptides with Gly as every third residue and a high imino acid content will spontaneously self-assemble into homotrimers with a triple-helical structure. Such stable homotrimer peptides have been well characterized in terms of stability, folding, and dynamics, and molecular structures have been obtained by NMR and X-ray crystallography. These homotrimer peptides serve as good models for collagen molecules with three identical chains, such as type Ⅱ collagen in cartilage. It has proved more difficult to obtain and characterize hybrid triple-helical peptides composed of chains with different amino acid sequences (heterotrimers), which can serve as models for heterotrimeric collagens such as type Ⅰ collagen in bone and type Ⅳ collagen in basement membranes. Heterotrimer peptide design strategies have included covalent linkage to force the selection of three chains and their alignment, while recent studies used electrostatic interactions within the (Gly-X-Y)_n sequences to direct desired self-assembly. Thus far, techniques for biophysical characterization of heterotrimers have probed average properties of the triple-helix. For instance, Gauba and Hartgerink used circular dichroism (CD) spectroscopy to follow the thermal stabilities of single peptide versus mixed peptide solutions, and differences in stability are interpreted in terms of homotrimer and heterotrimer molecules. In contrast, NMR has the capacity to follow the properties of individual residues and individual chains within trimers. Complexes where one subunit is labeled and others are not have been studied by NMR to define the features of the labeled complex and its interaction. Here, a similar strategy is applied to the triple-helix, using NMR to monitor labeled residues within one peptide chain to follow specific structural and chemical properties within a heterotrimer versus a homotrimer context.