Synthetic and structural studies on hydroxyproline-rich glycopeptides

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Proline is unique among the proteinogenic amino acids as its side chain is fused to the peptide backbone. As a consequence, there is a reduction in the energy difference between the prolyl amide cis and trans isomers, making them nearly isoenergetic; this leads to a higher cis N-terminal amide population relative to other amino acids. Moreover, the isomerization of the prolyl amide bond has been shown to be the rate-determining step in the folding pathway of many peptides and proteins. Besides exhibiting unique conformational properties and inducing important secondary structures (beta-turns and polyproline helix) proline also undergoes post-translational modifications such as hydroxylation followed sometimes by glycosylation. Glycosylation of hydroxyproline (Hyp) is widespread in the plant kingdom and occurs in Hyp-rich glycoprotein/peptides (HRGPs) that are associated with the cell walls of algae and flowering plants. However, the structural, conformational and biological implications of Hyp-glycosylation have not been addressed. This discovery grant explores the structure-function relationships of glycosylated Hyp-peptides in model peptides. In the short-term we will develop synthetic methodologies to prepare suitably protected glycosyl hydroxyproline building blocks that will be incorporated into peptides by solid-phase synthesis. In the longer-term, we will probe the structural, conformational, kinetic and physical properties of Hyp-glycosylation in plant-derived HRGPs as well as artificial Hyp-glycosylation of human-derived Hyp-rich peptides by nuclear magnetic resonance, circular dichroism and differential scanning calorimetry. The outcome of our research will provide for the first time insight into the structural, conformational, physical and biological implications of post-translational Hyp glycosylation in proteins and peptides and will impact many diverse research areas ranging from structural (glyco)biology to materials science.