title: Criteria for selecting PEGylation sites on proteins for higher thermodynamic and proteolytic stability.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, Brigham Young University , Provo, Utah 84602, United States.
authors.forename: Paul B
authors.initials: PB
authors.lastname: Lawrence
authors.forename: Yulian
authors.initials: Y
authors.lastname: Gavrilov
authors.forename: Sam S
authors.initials: SS
authors.lastname: Matthews
authors.forename: Minnie I
authors.initials: MI
authors.lastname: Langlois
authors.forename: Dalit
authors.initials: D
authors.lastname: Shental-Bechor
authors.forename: Harry M
authors.initials: HM
authors.lastname: Greenblatt
authors.forename: Brijesh K
authors.initials: BK
authors.lastname: Pandey
authors.forename: Mason S
authors.initials: MS
authors.lastname: Smith
authors.forename: Ryan
authors.initials: R
authors.lastname: Paxman
authors.forename: Chad D
authors.initials: CD
authors.lastname: Torgerson
authors.forename: Jacob P
authors.initials: JP
authors.lastname: Merrell
authors.forename: Cameron C
authors.initials: CC
authors.lastname: Ritz
authors.forename: Maxim B
authors.initials: MB
authors.lastname: Prigozhin
authors.forename: Yaakov
authors.initials: Y
authors.lastname: Levy
authors.forename: Joshua L
authors.initials: JL
authors.lastname: Price
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D008969:Molecular Sequence Data; D011092:Polyethylene Glycols / Q000737:chemistry*; D011487:Protein Conformation; D055550:Protein Stability; D011506:Proteins / Q000737:chemistry*; D013816:Thermodynamics
chemical_list: D011506:Proteins; D011092:Polyethylene Glycols