The bulky dehydroamino acids dehydrovaline (ΔVal) and dehydroethylnorvaline (ΔEnv) can be inserted into the turn regions of β-hairpin peptides without altering their secondary structures. These residues increase proteolytic stability, with ΔVal at the (i + 1) position having the most substantial impact. Additionally, a bulky dehydroamino acid can be paired with a d-amino acid (i.e., d-Pro) to synergistically enhance resistance to proteolysis. A link between proteolytic stability and peptide structure is established by the finding that a stabilized ΔVal-containing β-hairpin is more highly folded than its Asn-containing congener.

Anions have long been known to engage in stabilizing interactions with electron-deficient arenes. However, the precise nature and energetic contribution of anion−π interactions to protein stability remains a subject of debate. Here, we show that placing a negatively charged Asp in close proximity to electron-rich Phe in a reverse turn within the WW domain results in a favorable interaction that increases WW conformational stability by −1.3 kcal/mol.

The development of chemical strategies for site-specific protein modification now enables researchers to attach polyethylene glycol (PEG) to a protein drug at one or more specific locations (i.e., protein PEGylation). However, aside from avoiding enzyme active sites or protein-binding interfaces, distinguishing the optimal PEGylation site from the available alternatives has conventionally been a matter of trial and error. As part of a continuing effort to develop guidelines for identifying optimal PEGylation sites within proteins, we show here that the impact of PEGylation at various sites within the β-sheet model protein WW depends strongly on the identity of the PEG–protein linker. The PEGylation of Gln or of azidohomoalanine has a similar impact on WW conformational stability as does Asn–PEGylation, whereas the PEGylation of propargyloxyphenylalanine is substantially stabilizing at locations where Asn–PEGylation was destabilizing. Importantly, we find that at least one of these three site-specific PEGylation strategies leads to substantial PEG-based stabilization at each of the positions investigated, highlighting the importance of considering conjugation strategy as an important variable in selecting optimal PEGylation sites. We further demonstrate that using a branched PEG oligomer intensifies the impact of PEGylation on WW conformational stability and also show that PEG-based increases to conformational stability are strongly associated with corresponding increases in proteolytic stability.

Cooperative catalytic systems are making significant advances in modern organic synthesis due to the potential to combine multiple catalytic cycles or enable enzyme-like proximity effects. We report the rational design of a bifunctional helical peptide catalyst that displays an imidazolidinone catalyst in close proximity to a thiourea binding site and enables proximity-enhanced reactivity and selectivity. The helical structure of the peptide and the binding of both reactants are shown to be essential for enhanced reactivity in Diels–Alder and indole alkylation reactions, and up to 28 000 catalyst turnovers are achieved. A variety of Lewis basic functional groups facilitate binding and proximity-enhanced reactivity, and product selectivity is observed that cannot be achieved in the absence of the peptide template.Keywords: bifunctional peptide catalyst; Diels−Alder; proximity effects; thiourea binding site;

The interaction of a positively charged amino acid residue with a negatively charged residue (i.e. a salt bridge) can contribute substantially to protein conformational stability, especially when two ionic groups are in close proximity. At longer distances, this stabilizing effect tends to drop off precipitously. However, several lines of evidence suggest that salt-bridge interaction could persist at longer distances if an aromatic amino acid residue were positioned between the anion and cation. Here we explore this possibility in the context of a peptide in which a Lys residue occupies the i + 8 position relative to an i-position Glu on the solvent-exposed surface of a helix-bundle homotrimer. Variable temperature circular dichroism (CD) experiments indicate that an i + 4-position Trp enables a favorable long-range interaction between Glu and the i + 8 Lys. A substantial portion of this effect relies on the presence of a hydrogen-bond donor on the arene; however, non-polar arenes, a cyclic hydrocarbon, and an acyclic Leu side-chain can also enhance the long-range salt bridge, possibly by excluding water and ions from the space between Glu and Lys.

Site-specific PEGylation is an important strategy for enhancing the pharmacokinetic properties of protein drugs, and has been enabled by the recent development of many chemoselective reactions for protein side-chain modification. However, the impact of these different conjugation strategies on the properties of PEG–protein conjugates is poorly understood. Here we show that the ability of PEG to enhance protein conformational stability depends strongly on the identity of the PEG–protein linker, with the most stabilizing linkers involving conjugation of PEG to planar polar groups near the peptide backbone. We also find that branched PEGs provide superior stabilization relative to their linear counterparts, suggesting additional applications for branched PEGs in protein stabilization.

PEGylation of protein side chains has been used for more than 30 years to enhance the pharmacokinetic properties of protein drugs. However, there are no structure- or sequence-based guidelines for selecting sites that provide optimal PEG-based pharmacokinetic enhancement with minimal losses to biological activity. We hypothesize that globally optimal PEGylation sites are characterized by the ability of the PEG oligomer to increase protein conformational stability; however, the current understanding of how PEG influences the conformational stability of proteins is incomplete. Here we use the WW domain of the human protein Pin 1 (WW) as a model system to probe the impact of PEG on protein conformational stability. Using a combination of experimental and theoretical approaches, we develop a structure-based method for predicting which sites within WW are most likely to experience PEG-based stabilization, and we show that this method correctly predicts the location of a stabilizing PEGylation site within the chicken Src SH3 domain. PEG-based stabilization in WW is associated with enhanced resistance to proteolysis, is entropic in origin, and likely involves disruption by PEG of the network of hydrogen-bound solvent molecules that surround the protein. Our results highlight the possibility of using modern site-specific PEGylation techniques to install PEG oligomers at predetermined locations where PEG will provide optimal increases in conformational and proteolytic stability.

PEGylation is an important strategy for enhancing the pharmacokinetic properties of protein drugs. Modern chemoselective reactions now enable specific placement of a single PEG at any site on a protein surface. However, few rational structure-based guidelines exist for selecting optimal PEGylation sites. Here, we explore the impact of PEGylation on the conformational stability of α-helices using an α-helical coiled coil as a model system. We find that maleimide-based PEGylation of a solvent-exposed i position Cys can stabilize coiled-coil quaternary structure when Lys residues occupy both the i + 3 and i + 4 positions, due to favorable interactions between the PEG-maleimide and the Lys residues. Applying this Cysi–Lysi+3–Lysi+4 triad to a solvent-exposed position within the C-terminal helix of the villin headpiece domain leads to similar PEG-based increases in conformational stability, highlighting the possibility of using the Cysi–Lysi+3–Lysi+4 triad as a general strategy for PEG-based stabilization of helical proteins.

PEGylation, or addition of poly(ethylene glycol) chains to proteins, is widely used to improve delivery in pharmaceutical applications. Recent studies suggest that stabilization of a protein by PEG, and hence its proteolytic degradability, is sequence-dependent and requires only short PEG chains. Here we connect stabilization by short PEG chains directly to the structural dynamics of the protein and PEG chain. We measured the stability of human Pin1 WW domain with PEG-4 at asparagine 19 for a full mutant cycle at two positions thought to influence PEG–protein interaction: Ser16Ala and Tyr23Phe. We then performed explicit solvent molecular dynamics simulations on all PEGylated and PEG-free mutants. The mutant cycle yields a nonadditive stabilization effect where the pseudo-wild type and double mutant are more stabilized relative to unPEGylated proteins than are the two single mutants. The simulation reveals why: the double mutant suffers loss of β-sheet structure, which PEGylation restores even though the PEG extends as a coil into the solvent. In contrast, in one of the single mutants, PEG preferentially interacts with the protein surface while disrupting the interactions of its asparagine host with a nearby methionine side chain. Thus, PEG attachment can stabilize a protein differentially depending on the local sequence, and either by interacting with the surface or by extending into the solvent. A simulation with PEG-45 attached to asparagine 19 shows that PEG even can do both in the same context.

N-glycans play important roles in many cellular processes and can increase protein conformational stability in specific structural contexts. Glycosylation (with a single GlcNAc) of the reverse turn sequence Phe-Yyy-Asn-Xxx-Thr at Asn stabilizes the Pin 1 WW domain by −0.85 ± 0.12 kcal mol–1. Alternative methods exist for attaching carbohydrates to proteins; some occur naturally (e.g., the O-linkage), whereas others use chemoselective ligation reactions to mimic the natural N- or O-linkages. Here, we assess the energetic consequences of replacing the Asn linkage in the glycosylated WW domain with a Gln linkage, with two natural O-linkages, with two unnatural triazole linkages, and with an unnatural α-mercaptoacetamide linkage. Of these alternatives, only glycosylation of the triazole linkages stabilizes WW, and by a smaller amount than N-glycosylation, highlighting the need for caution when using triazole- or α-mercaptoacetamide-linked carbohydrates to mimic native N-glycans, especially where the impact of glycosylation on protein conformational stability is important.

Protein PEGylation is an effective method for reducing the proteolytic susceptibility, aggregation propensity, and immunogenicity of protein drugs. These pharmacokinetic challenges are fundamentally related to protein conformational stability, and become much worse for proteins that populate the unfolded state under ambient conditions. If PEGylation consistently led to increased conformational stability, its beneficial pharmacokinetic effects could be extended and enhanced. However, the impact of PEGylation on protein conformational stability is currently unpredictable. Here we show that appending a short PEG oligomer to a single Asn side chain within a reverse turn in the WW domain of the human protein Pin 1 increases WW conformational stability in a manner that depends strongly on the length of the PEG oligomer: shorter oligomers increase folding rate, whereas longer oligomers increase folding rate and reduce unfolding rate. This strong length dependence is consistent with the possibility that the PEG oligomer stabilizes the transition and folded states of WW relative to the unfolded state by interacting favorably with side-chain or backbone groups on the WW surface.

The interaction of a positively charged amino acid residue with a negatively charged residue (i.e. a salt bridge) can contribute substantially to protein conformational stability, especially when two ionic groups are in close proximity. At longer distances, this stabilizing effect tends to drop off precipitously. However, several lines of evidence suggest that salt-bridge interaction could persist at longer distances if an aromatic amino acid residue were positioned between the anion and cation. Here we explore this possibility in the context of a peptide in which a Lys residue occupies the i + 8 position relative to an i-position Glu on the solvent-exposed surface of a helix-bundle homotrimer. Variable temperature circular dichroism (CD) experiments indicate that an i + 4-position Trp enables a favorable long-range interaction between Glu and the i + 8 Lys. A substantial portion of this effect relies on the presence of a hydrogen-bond donor on the arene; however, non-polar arenes, a cyclic hydrocarbon, and an acyclic Leu side-chain can also enhance the long-range salt bridge, possibly by excluding water and ions from the space between Glu and Lys.