N-glycosylation of eukaryotic proteins helps them fold and traverse the cellular secretory pathway and can increase their stability, although the molecular basis for stabilization is poorly understood. Glycosylation of proteins at naïve sites (ones that normally are not glycosylated) could be useful for therapeutic and research applications, but currently results in unpredictable changes to protein stability. We show that placing a Phe residue two or three positions prior to a glycosylated Asn in distinct reverse turns facilitates stabilizing interactions between the aromatic side chain and the first N-acetylglucosamine (GlcNAc) of the glycan. Glycosylating this portable structural module, an “enhanced aromatic sequon”, in three different proteins stabilizes their native states by −0.7 to −2.0 kilocalories per mole and increases cellular glycosylation efficiency.

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Summary Signaling in the most conserved branch of the endoplasmic reticulum (ER) unfolded protein response (UPR) is initiated by sequence-specific cleavage of the HAC1/XBP1 mRNA by the ER stress-induced kinase-endonuclease IRE1. We have discovered that the flavonol quercetin activates yeast IRE1’s RNase and potentiates activation by ADP, a natural activating ligand that engages the IRE1 nucleotide binding cleft. Enzyme kinetics and the structure of a co-crystal of IRE1 complexed with ADP and quercetin reveal engagement by quercetin of an unanticipated ligand binding pocket at the dimer interface of IRE1’s Kinase Extension Nuclease (KEN) domain. Analytical ultracentrifugation and crosslinking studies support the preeminence of enhanced dimer formation in quercetin’s mechanism of action. These findings hint at the existence of endogenous cytoplasmic ligands that may function alongside stress signals from the ER lumen to modulate IRE1 activity and at the potential for the development of drugs that modify UPR signaling from this unanticipated site.

The function of a protein generally depends on adoption of a specific folding pattern, which in turn is determined by the side chain sequence along the polypeptide backbone. Here we show that the sequence-encoded structural information in peptides derived from yeast transcriptional activator GCN4 can be used to prepare hybrid α/β-peptide foldamers that adopt helix bundle quaternary structures. Crystal structures of two hybrid α/β-peptides are reported along with detailed structural comparison to α-peptides of analogous side chain sequence. There is considerable homology between α- and α/β-peptides at the level of helical secondary structure, with modest but significant differences in the association geometry of helices in the quaternary structure.

The extent to which polypeptide conformation depends on side-chain composition and sequence has been widely studied, but less is known about the importance of maintaining an α-amino acid backbone. Here, we examine a series of peptides with backbones that feature different repeating patterns of α- and β-amino acid residues but an invariant side-chain sequence. In the pure α-backbone, this sequence corresponds to the previously studied peptide GCN4-pLI, which forms a very stable four-helix bundle quaternary structure. Physical characterization in solution and crystallographic structure determination show that a variety of α/β-peptide backbones can adopt sequence-encoded quaternary structures similar to that of the α prototype. There is a loss in helix bundle stability upon β-residue incorporation; however, stability of the quaternary structure is not a simple function of β-residue content. We find that cyclically constrained β-amino acid residues can stabilize the folds of α/β-peptide GCN4-pLI analogues and restore quaternary structure formation to backbones that are predominantly unfolded in the absence of cyclic residues. Our results show a surprising degree of plasticity in terms of the backbone compositions that can manifest the structural information encoded in a sequence of amino acid side chains. These findings offer a framework for the design of nonnatural oligomers that mimic the structural and functional properties of proteins.