The "side chain" from the α carbon connects to the nitrogen forming a pyrrolidine loop, classifying it as a aliphatic amino acid.

Proline is the only proteinogenic amino acid which is a secondary amine, as the nitrogen atom is attached both to the α-carbon and to a chain of three carbons that together form a five-membered ring.

[9][10][11] In plants, proline accumulation is a common physiological response to various stresses but is also part of the developmental program in generative tissues (e.g.

Peptide bond formation with incoming Pro-tRNAPro in the ribosome is considerably slower than with any other tRNAs, which is a general feature of N-alkylamino acids.

The hydroxylation of proline by prolyl hydroxylase (or other additions of electron-withdrawing substituents such as fluorine) increases the conformational stability of collagen significantly.

Severe diseases such as scurvy can result from defects in this hydroxylation, e.g., mutations in the enzyme prolyl hydroxylase or lack of the necessary ascorbate (vitamin C) cofactor.

Peptide bonds to proline, and to other N-substituted amino acids (such as sarcosine), are able to populate both the cis and trans isomers.

By contrast, the cis and trans isomers of the X-Pro peptide bond (where X represents any amino acid) both experience steric clashes with the neighboring substitution and have a much lower energy difference.

[21] However, these values depend on the preceding amino acid, with Gly[22] and aromatic[23] residues yielding increased fractions of the cis isomer.

Due to the ring formation connected to the beta carbon, the ψ and φ angles about the peptide bond have fewer allowable degrees of rotation.

Additionally, proline is the only amino acid that does not form a red-purple colour when developed by spraying with ninhydrin for uses in chromatography.