Effects of glycosylation of (2S,4R)-4-hydroxyproline on the conformation, kinetics, and thermodynamics of prolyl amide isomerization.

Glycosylation is a common post-translational modification of proteins implicated in cellular recognition processes and controlling protein conformation.1 Typically, carbohydrates are O-linked to serine (Ser) and threonine (Thr) or N-linked to asparagine (Asn). Glycosylation of (2S,4R)-4-hydroxyproline (Hyp) is widespread in the plant kingdom and occurs in Hyp-rich glycoproteins (HRGPs) that are associated with the cell walls of algae and flowering plants.2 HRGPs are characterized by extensively glycosylated Hyp sequences that contain O-glycosidic linkages to the pyranose Dgalactose or the furanose L-arabinose.2 Although HRGPs are broadly implicated in many aspects of plant growth, development3 and cell wall stability,2 no information is available about the structural and conformational implications of Hyp-glycosylation on peptide backbone conformation. Hyp and proline (Pro) are unique among the proteinogenic amino acids since they are characterized by limited rotation of the ø dihedral angle (Figure 1) as their side chain is fused to the peptide backbone. As a consequence, there is a reduction in the energy difference between the prolyl amide cis and trans isomers, making them nearly isoenergetic; this leads to higher cis N-terminal amide content relative to the other amino acids. Moreover, the isomerization of the prolyl amide bond has been shown to be the ratedetermining step in the folding pathways of many peptides and proteins.4 Herein we describe the effects of galactosylation of Hyp on the conformation as well as the thermodynamics and kinetics of prolyl N-terminal amide isomerization. Compounds 4a AcHyp(R-D-Gal)NHMe and 4b AcHyp(â-D-Gal)NHMe were selected as glycopeptide mimics, while AcProNHMe 1, AcHypNHMe 2, and AcHyp(Otert-butyl)NHMe 3 served as non-glycosylated reference compounds (Figure 1). The trans rotamers in compounds 1-4b were assigned on the basis of higher Cδ atom NMR chemical shifts relative to the cis rotamer5 and nOe transfer between H-δ of proline and the N-acyl protons in selective 1D GOESY experiments.6 The relative amounts of cis and trans isomers were determined by integrating and averaging as many distinct proton signals as possible for both the major and minor isomers in the 1H NMR spectra.7 In D2O at 37 °C, the trans/cis isomer ratio equilibrium constant (Kt/c) for 4a (3.41 ( 0.30) and 4b (3.37 ( 0.28) are nearly identical to those of 2 (3.52 ( 0.05) and 3 (3.34 ( 0.15), and the observed differences are within the experimental errors (Table 3). The kinetics of cis/trans isomerization for compounds 1-4b were determined by 1H NMR spectroscopy inversion transfer experiments8 in D2O at elevated temperature. At 67 °C, the cis-to-trans rate constant of isomerization (kct) of the R-glycosylated Hyp model peptide 4a (kct ) 0.83 s-1) is very similar compared to the hydroxyproline model peptide 2 (kct of 0.73 s-1) and 3 (kct ) 0.77 s-1), while the â-anomer 4b gave slightly lower rates (kct ) 0.61 s-1). A similar trend was observed for the trans-to-cis rate constants of compounds 1-4b (Table 1). At physiological temperature the kinetic rates are too slow to be differentiated by this assay. The effects of temperature on kct and ktc were analyzed by Eyring plots9 (Supporting Information) and values for ∆H‡ and ∆S‡ were calculated from linear least-squares fits of the data in these plots and are presented in Table 2. The activation parameters demonstrate that the free-energy barriers to isomerization of compounds 1-4b Figure 1. Cis-trans isomerization of reference diamides 1-3 and the galactosylated Hyp model amides 4a and 4b.