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1.
Biochemistry ; 60(26): 2064-2070, 2021 07 06.
Article in English | MEDLINE | ID: mdl-34137579

ABSTRACT

Here we show that an NH-π interaction between a highly conserved Asn and a nearby Trp stabilizes the WW domain of the human protein Pin1. The strength of this NH-π interaction depends on the structure of the arene, with NH-π interactions involving Trp or naphthylalanine being substantially more stabilizing than those involving Tyr or Phe. Calculations suggest arene size and polarizability are key structural determinants of NH-π interaction strength. Methylation or PEGylation of the Asn side-chain amide nitrogen each strengthens the associated NH-π interaction, though likely for different reasons. We hypothesize that methylation introduces steric clashes that destabilize conformations in which the NH-π interaction is not possible, whereas PEGylation strengthens the NH-π interaction via localized desolvation of the protein surface.


Subject(s)
Asparagine/chemistry , Hydrogen Bonding/drug effects , NIMA-Interacting Peptidylprolyl Isomerase/chemistry , Polyethylene Glycols/chemistry , Tryptophan/chemistry , WW Domains/drug effects , Amino Acid Sequence , Humans , Methylation , Models, Molecular , Mutation , NIMA-Interacting Peptidylprolyl Isomerase/genetics , Protein Conformation , Thermodynamics , WW Domains/genetics
2.
J Org Chem ; 85(3): 1725-1730, 2020 02 07.
Article in English | MEDLINE | ID: mdl-31749365

ABSTRACT

Many proteins have one or more surface-exposed patches of nonpolar residues; our observations here suggest that PEGylation near such locations might be a useful strategy for increasing protein conformational stability. Specifically, we show that conjugating a PEG-azide to a propargyloxyphenylalanine via the copper(I)-catalyzed azide-alkyne cycloaddition can increase the conformational stability of the WW domain due to a favorable synergistic effect that depends on the hydrophobicity of a nearby patch of nonpolar surface residues.


Subject(s)
Polyethylene Glycols , Proteins , Alkynes , Azides , Copper , Protein Conformation , Protein Stability , WW Domains
3.
ACS Chem Biol ; 14(7): 1652-1659, 2019 07 19.
Article in English | MEDLINE | ID: mdl-31188563

ABSTRACT

Conjugation of polyethylene glycol (PEGylation) is a well-known strategy for extending the serum half-life of protein drugs and for increasing their resistance to proteolysis and aggregation. We previously showed that PEGylation can increase protein conformational stability; the extent of PEG-based stabilization depends on the PEGylation site, the structure of the PEG-protein linker, and the ability of PEG to release water molecules from the surrounding protein surface to the bulk solvent. The strength of a noncovalent interaction within a protein depends strongly on its microenvironment, with salt-bridge and hydrogen-bond strength increasing in nonpolar versus aqueous environments. Accordingly, we wondered whether partial desolvation by PEG of the surrounding protein surface might result in measurable increases in the strength of a salt bridge near a PEGylation site. Here we explore this possibility using triple-mutant box analysis to assess the impact of PEGylation on the strength of nearby salt bridges at specific locations within three peptide model systems. The results indicate that PEG can increase the nearby salt-bridge strength, though this effect is not universal, and its precise structural prerequisites are not a simple function of secondary structural context, of the orientation and distance between the PEGylation site and salt bridge, or of salt-bridge residue identity. We obtained high-resolution X-ray diffraction data for a PEGylated peptide in which PEG enhances the strength of a nearby salt bridge. Comparing the electron density map of this PEGylated peptide versus that of its non-PEGylated counterpart provides evidence of localized protein surface desolvation as a mechanism for PEG-based salt-bridge stabilization.


Subject(s)
Peptides/chemistry , Polyethylene Glycols/chemistry , Proteins/chemistry , Salts/chemistry , Databases, Protein , Models, Molecular , Protein Aggregates , Protein Conformation , Protein Folding , Protein Stability , Proteolysis
4.
Org Biomol Chem ; 16(46): 8933-8939, 2018 11 28.
Article in English | MEDLINE | ID: mdl-30444518

ABSTRACT

Hydrocarbon stapling and PEGylation are distinct strategies for enhancing the conformational stability and/or pharmacokinetic properties of peptide and protein drugs. Here we combine these approaches by incorporating asparagine-linked O-allyl PEG oligomers at two positions within the ß-sheet protein WW, followed by stapling of the PEGs via olefin metathesis. The impact of stapling two sites that are close in primary sequence is small relative to the impact of PEGylation alone and depends strongly on PEG length. In contrast, stapling of two PEGs that are far apart in primary sequence but close in tertiary structure provides substantially more stabilization, derived mostly from an entropic effect. Comparison of PEGylation + stapling vs. alkylation + stapling at the same positions in WW reveals that both approaches provide similar overall levels of conformational stability.


Subject(s)
Asparagine/analogs & derivatives , Entropy , Peptides/chemistry , Polyethylene Glycols/chemistry , Proteins/chemistry , Alkenes/chemistry , Models, Molecular , Protein Conformation , Protein Conformation, beta-Strand , Protein Stability , WW Domains
5.
Bioconjug Chem ; 28(10): 2507-2513, 2017 10 18.
Article in English | MEDLINE | ID: mdl-28972368

ABSTRACT

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 ß-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.


Subject(s)
Polyethylene Glycols/chemistry , Proteins/chemistry , Proteins/metabolism , Proteolysis , Amino Acid Sequence , Models, Molecular , Protein Conformation, beta-Strand , Protein Stability
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