Context-Dependent Stabilizing Interactions among Solvent-Exposed Residues along the Surface of a Trimeric Helix Bundle.

Here we show that a solvent-exposed -position (i.e., residue 14) within a well-characterized trimeric helix bundle can facilitate a stabilizing long-range synergistic interaction involving -position Glu10 (i.

Incorporation of Aza-Glycine into Collagen Peptides.

Substitution of natural amino acids with their aza-amino acid counterparts in peptides has been a historically challenging prospect due to the diminished reactivity of the involved reagents. Current methods require lengthy reaction times or difficult synthetic strategies. Aza-glycine has proven to be a valuable tool in the design of triple-helix-forming collagen peptides.

Influence of PEGylation on the Strength of Protein Surface Salt Bridges.

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.

Stapling of two PEGylated side chains increases the conformational stability of the WW domain via an entropic effect.

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 β-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.

Bulky Dehydroamino Acids Enhance Proteolytic Stability and Folding in β-Hairpin Peptides.

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.

An Anion-π Interaction Strongly Stabilizes the β-Sheet Protein WW.

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.

Enhancing a long-range salt bridge with intermediate aromatic and nonpolar amino acids.

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.

Criteria for selecting PEGylation sites on proteins for higher thermodynamic and proteolytic stability.

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.

Cys(i)-Lys(i+3)-Lys(i+4) triad: a general approach for PEG-based stabilization of α-helical proteins.

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.

Impact of site-specific PEGylation on the conformational stability and folding rate of the Pin WW domain depends strongly on PEG oligomer length.

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.