4,4-Difluoroproline as a Unique F NMR Probe of Proline Conformation.

Despite the importance of proline conformational equilibria ( versus amide and versus ring pucker) on protein structure and function, there is a lack of convenient ways to probe proline conformation. 4,4-Difluoroproline (Dfp) was identified to be a sensitive F NMR-based probe of proline conformational biases and isomerism. Within model compounds and disordered peptides, the diastereotopic fluorines of Dfp exhibit similar chemical shifts (Δδ = 0-3 ppm) when a X-Dfp amide bond is present. In contrast, the diastereotopic fluorines exhibit a large (Δδ = 5-12 ppm) difference in chemical shift in a X-Dfp prolyl amide bond. DFT calculations, X-ray crystallography, and solid-state NMR spectroscopy indicated that Δδ directly reports on the relative preference of one proline ring pucker over the other: a fluorine which is pseudo-axial (i.e., the pro-4-F in an ring pucker, or the pro-4-F in an ring pucker) is downfield, while a fluorine which is pseudo-equatorial (i.e., pro-4-F when , or pro-4-F when ) is upfield. Thus, when a proline is disordered (a mixture of and ring puckers, as at -Pro in peptides in water), it exhibits a small Δδ. In contrast, when the Pro is ordered (i.e., when one ring pucker is strongly preferred, as in -Pro amide bonds, where the ring pucker is strongly favored), a large Δδ is observed. Dfp can be used to identify inherent induced order in peptides and to quantify proline isomerism. Using Dfp, we discovered that the stable polyproline II helix (PPII) formed in the denatured state (8 M urea) exhibits essentially equal populations of the and proline ring puckers. In addition, the data with Dfp suggested the specific stabilization of PPII by water over other polar solvents. These data strongly support the importance of carbonyl solvation and n → π* interactions for the stabilization of PPII. Dfp was also employed to quantify proline isomerism as a function of phosphorylation and the R406W mutation in peptides derived from the intrinsically disordered protein tau. Dfp is minimally sterically disruptive and can be incorporated in expressed proteins, suggesting its broad application in understanding proline isomerization, protein folding, and local order in intrinsically disordered proteins.