The hydroxyproline-rich glycoproteins (HRGPs) are the major structural proteins of the extracellular matrix of algae and land plants. They are characterized by a rigid polyproline type II (PPII) conformation and extensive O-glycosylation of 4(R)-hydroxy-l-proline (Hyp) residues, which is a unique post-translational modification of proteins. The functional consequences of HRGP glycosylation remains unclear, but they have been implicated in contributing to their structural rigidity. Here, we have investigated the effects of naturally occurring beta-O-galactosylation of Hyp residues on the conformational stability of the PPII helix. In a series of well-defined model peptides Ac-(l-proline)(9)-NH(2) (1), Ac-(Hyp)(9)-NH(2) (2), and Ac-[Hyp(beta-d-galactose)](9)-NH(2) (3) we demonstrate that contiguous O-glycosylation of Hyp residues causes a dramatic increase in the thermal stability of the PPII helix according to analysis of thermal melting curves. This represents the first quantitative data on the contributions of glycosylation to stabilizing the PPII conformation. Molecular modeling indicates the increase in conformational stability may be due to a regular network of interglycan and glycan-peptide hydrogen bonds, in which the carbohydrate residues form a hydrophilic "overcoat" of the PPII helix. Evidence of this shielding effect of the amide backbone may be provided by analysis of the circular dichroism bands, which indicates an increase in the rho value of 3 relative to 1 and 2. This study gives further insight into the effects of naturally occurring Hyp beta-O-linked glycans on the PPII conformation as found in HRGPs in plant cell walls and also indicates that polyproline sequences may be suitable for the development of molecular scaffolds for the presentation of glycan structures.

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, development 3 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( -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 C δ atom NMR chemical shifts relative to the cis rotamer 5 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 1 H NMR spectra. 7 In D 2 O at 37°C, the trans/cis isomer ratio equilibrium constant (K t/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 1 H NMR spectroscopy inversion transfer experiments 8 in D 2 O at elevated temperature. At 67°C, the cis-to-trans rate constant of isomerization (k ct ) of the R-glycosylated Hyp model peptide 4a (k ct ) 0.83 s -1 ) is very similar compared to the hydroxyproline model peptide 2 (k ct of 0.73 s -1 ) and 3 (k ct ) 0.77 s -1 ), while the -anomer 4b gave slightly lower rates (k ct ) 0.61 s -1 ). A similar trend was observed for the trans-to-cis rate const...

The conformations of peptides and proteins are often influenced by glycans O-linked to serine (Ser) or threonine (Thr). (2S,4R)-4-Hydroxyproline (Hyp), together with L-proline (Pro), are interesting targets for O-glycosylation because they have a unique influence on peptide and protein conformation. In previous work we found that glycosylation of Hyp does not affect the N-terminal amide trans/cis ratios (K(trans/cis)) or the rates of amide isomerization in model amides. The stereoisomer of Hyp--(2S,4S)-4-hydroxyproline (hyp)--is rarely found in nature, and has a different influence both on the conformation of the pyrrolidine ring and on K(trans/cis). Glycans attached to hyp would be expected to be projected from the opposite face of the prolyl side chain relative to Hyp; the impact this would have on K(trans/cis) was unknown. Measurements of (3)J coupling constants indicate that the glycan has little impact on the C(gamma)-endo conformation produced by hyp. As a result, it was found that the D-galactose residue extending from a C(gamma)-endo pucker affects both K(trans/cis) and the rate of isomerization, which is not found to occur when it is projected from a C(gamma)-exo pucker; this reflects the different environments delineated by the proline side chain. The enthalpic contributions to the stabilization of the trans amide isomer may be due to disruption of intramolecular interactions present in hyp; the change in enthalpy is balanced by a decrease in entropy incurred upon glycosylation. Because the different stereoisomers--Hyp and hyp--project the O-linked carbohydrates in opposite spatial orientations, these glycosylated amino acids may be useful for understanding of how the projection of a glycan from the peptide or protein backbone exerts its influence.

We describe the synthesis of a fused bicyclic C-glucosylproline hybrid (GlcProH) from commercially available 2,3,4,6-tetra-O-benzyl-d-glucopyranose. The GlcProH was incorporated into the model peptides Ac-GlcProH-NHMe and Ac-Gly-GlcProH-NHMe. Postsynthetic modifications can be introduced via derivatization of the carbohydrate scaffold. Conformational analysis of the GlcProH-modified model peptides shows that while the conformation of GlcProH remains fixed, the prolyl N-terminal amide equilibrium (Kt/c) can be varied with different modifications of the carbohydrate scaffold. Simple N-acyl derivatives studied by NMR spectroscopy showed that in CD3OD there was an increase in the cis-amide content as the sugar substituents changed from benzyl (10%) to hydroxyl (22%) to acetate (36%). Similar effects were observed in DMSO-d6. The exact nature of the influence is unclear, but it most likely arises through intramolecular interactions between sugar groups and the peptidic amide backbone. Overall, our GlcProH demonstrates variation in Kt/c through tuning of the carbohydrate scaffold: a new concept in proline peptidomimetics.

The structure of fused C-glucosylproline hybrid (GlcProH) has been studied in detail computationally. A systematic molecular mechanics/Monte Carlo search has been performed in order to cover the entire conformational space of GlcProH. This has been followed by density-functional (DFT B3LYP) calculations in the gas phase and in aqueous solution, using the polarizable continuum model (PCM). In the gas phase, a large excess of the cis conformation with respect to the prolyl amide bond is found. This is reversed in aqueous solution where the calculations show 80% trans conformers, which is in accordance with experimental data. Thus, the PCM model is capable of accurately predicting cis-trans ratios. The free energy of solvation is not correlated with the dipole moment. Hence, a model (such as PCM) is required that takes into account the complete charge distribution. The reversal of the cis-trans ratio between gas phase and solution also emphasizes the effects of different free energies of solvation for the distinct conformers. Nevertheless, the energy difference between the cis and trans conformers is very small in solution (0.18 kcal/mol). Intramolecular hydrogen bonding is found to stabilize the cis conformers exclusively, which is a result of the rigid geometry of the fused rings. This can be contrasted to related more flexible molecules that show hydrogen bonding for both cis and trans isomers. The hydrogen bonding is at least partially responsible for the preferential stabilization of the cis conformers in the gas phase and a very small cis-trans energy difference in solution.