Prolyl 4-hydroxylases install a hydroxyl group in the 4R configuration on the gamma-carbon atom of certain (2S)-proline (Pro) residues in tropocollagen, elastin, and other proteins to form (2S,4R)-4-hydroxyproline (Hyp). The gauche effect arising from this prevalent post-translational modification enforces a C-gamma-exo ring pucker and stabilizes the collagen triple helix. The Hyp diastereomer (2S,4S)-4-hydroxyproline (hyp) has not been observed in a protein, despite the ability of electronegative 4S substituents to enforce the more common C-gamma-endo ring pucker of Pro. Here, we use density functional theory, spectroscopy, crystallography, and calorimetry to explore the consequences of hyp incorporation on protein stability using a collagen model system. We find that the 4S-hydroxylation of Pro to form hyp does indeed enforce a C-gamma-endo ring pucker but a transannular hydrogen bond between the hydroxyl moiety and the carbonyl of hyp distorts the main-chain torsion angles that typically accompany a C-gamma-endo ring pucker. This same transannular hydrogen bond enhances an n ->pi* interaction that stabilizes the trans conformation of the peptide bond preceding hyp, endowing hyp with the unusual combination of a C-gamma-endo ring pucker and high trans/cis ratio. O-Methylation of hyp to form (2S,4S)-4-methoxyproline (mop) eliminates the transannular hydrogen bond and restores a prototypical C-gamma-endo pucker. mop residues endow the collagen triple helix with much more conformational stability than do hyp residues. These findings highlight the critical importance of the configuration of the hydroxyl group installed on C-gamma of proline residues.