College Lecturer/Assistant Professor
University College Dublin
The Impact of Fluoroproline Isomers on the Folding and Thermodynamic Stability of Globular Proteins
4-substituted proline analogues are of particular interest for investigating protein folding kinetics and thermodynamic stability, as substituents at the Cγ atom of proline have a strong effect on the two main conformational equilibria of proline residues in polypeptides, namely the endo/exo ring puckering of the proline ring and the cis/trans equilibrium of prolyl peptide bonds. The installing of electron-withdrawing substituents on the pyrrolydine ring shows to elicit a predictable effect on coiled-coil peptides. However, their impact in the context of the tertiary structure of globular proteins is contradictory.
We have analyzed the influence of (4R)- and (4S)–fluoroproline (Flp) on the thermodynamic stability and on the rate–limiting trans–to–cis isomerization of the Ile75–Pro76 peptide bond in the folding of Trx1P, an Escherichia coli thioredoxin (Trx) variant. We showed that both Flp isomers adopted the endo pucker in the tertiary structure of Trx1P and that both exert a similar effect on the thermodynamic stability of the protein.1 However, while (4R)–Flp at position 76 had no effect on the isomerization rate in the context of the intact tertiary structure, (4S)–Flp accelerated the folding reaction ninefold. Similarly, tenfold faster trans–to–cis isomerization of Ile75–(4S)–Flp76 relative to Ile75–Pro76 was observed in the unfolded state.2 Our results show that the replacement of cis prolines by non–natural proline analogues can be used for modulating the kinetics of the rate–limiting folding step of proteins with cis proly–peptide bonds in the native state.