The implications of (2S,4S)-hydroxyproline 4-O-glycosylation for prolyl amide isomerization

The conformations of peptides and proteins are often influenced by glycans O-linked to serine (Ser) or threonine (Thr). (2S,4R)-4-Hydroxyproline (Hyp), together with L-proline (Pro), are interesting targets for O-glycosylation because they have a unique influence on peptide and protein conformation. In previous work we found that glycosylation of Hyp does not affect the N-terminal amide trans/cis ratios (K(trans/cis)) or the rates of amide isomerization in model amides. The stereoisomer of Hyp--(2S,4S)-4-hydroxyproline (hyp)--is rarely found in nature, and has a different influence both on the conformation of the pyrrolidine ring and on K(trans/cis). Glycans attached to hyp would be expected to be projected from the opposite face of the prolyl side chain relative to Hyp; the impact this would have on K(trans/cis) was unknown. Measurements of (3)J coupling constants indicate that the glycan has little impact on the C(gamma)-endo conformation produced by hyp. As a result, it was found that the D-galactose residue extending from a C(gamma)-endo pucker affects both K(trans/cis) and the rate of isomerization, which is not found to occur when it is projected from a C(gamma)-exo pucker; this reflects the different environments delineated by the proline side chain. The enthalpic contributions to the stabilization of the trans amide isomer may be due to disruption of intramolecular interactions present in hyp; the change in enthalpy is balanced by a decrease in entropy incurred upon glycosylation. Because the different stereoisomers--Hyp and hyp--project the O-linked carbohydrates in opposite spatial orientations, these glycosylated amino acids may be useful for understanding of how the projection of a glycan from the peptide or protein backbone exerts its influence.