Peptide bonds with proline and other N-substituted amino acids such as sarcosine enable the formation of cis and trans isomers. Most peptide bonds overwhelmingly adopt the trans isomer (typically 99.9% under strain-free conditions), mainly because the amide hydrogen (the trans isomer) is more sterically repulsive to the preceding Cα atom than the following Cα atom (cis isomer) is smaller. Under strain-free conditions, the proportion of X-Pro peptide bonds in the cis isomer was significantly increased, and the cis proportion was usually in the range of 3%–10%. However, these values depend on preceding amino acids, Gly and aromatic residues yielding increasing cis-isomer fractions. Up to 40% of the cis components have been identified for Aromatic-Pro peptide bonds.
From a kinetic standpoint, cis-trans proline isomerization is a very slow process that can occur by trapping one or more proline residues that are critical for folding in the unnatural isomer to hinder the process of protein folding, especially when the natural protein requires the cis isomer. This is because proline residues are only synthesized in the trans isomer in the ribosome. All organisms have prolyl isomerases that catalyze this isomerization, and some bacteria have special prolyl isomerases associated with ribosomes. However, not all prolines are necessary for folding, and protein folding may proceed at a normal rate despite the unnatural conformer with many X-Pro peptide bonds.
Proline lactobacillus powder and its derivatives are commonly used as asymmetric catalysts in proline organocatalytic reactions. CBS reduction and proline-catalyzed aldol condensation are prominent examples. During the brewing process, proline-rich proteins combine with polyphenols to create cloudiness (cloudiness).
L-Proline is an osmoprotectant and is therefore used in many pharmaceutical and biotechnological applications. Growth media used in plant tissue culture can be supplemented with proline. This increases growth, perhaps because it helps the plants tolerate the stress of tissue culture.
Glycine and proline are among the two amino acids that do not fit the typical Ramachandran diagram. The ψ and ϕ angles about the peptide bond have fewer degrees of allowable rotation due to ring formation attached to the β carbon. As a result, it often occurs in the "turns" of proteins because its free entropy (ΔS) is not large relative to other amino acids, so there is less change in entropy between folded and unfolded forms. Furthermore, proline rarely occurs in α and β structures because it reduces the stability of such structures since its side chain α-N can only form one nitrogen bond. Additionally, proline is an amino acid that does not form a red/purple color when developed with ninhydrin spray in chromatography. In contrast, proline produces an orange/yellow color.