Analysis of MonoPEGylated Human Galectin-2 by Small-Angle X-ray and Neutron Scattering: Concentration Dependence of PEG Conformation in the Conjugate

Abstract

Protein conjugation with polyethylene glycol (PEG) is a valuable means for improving stability, solubility, and bioavailability of pharmaceutical proteins. Using human galectin-2 (hGal-2) and 5 kDa PEG as a model system we first produced a PEG-hGal-2 conjugate exclusively at the Cys75 residue, resulting in two monosubstituted subunits per hGal-2 homodimer. Small angle X-ray and neutron scattering (SAXS and SANS) were combined to provide complementary structural information about the PEG-hGal-2 conjugate, wherein signal generation in SAXS depends mainly on the protein while SANS data presents signals from both the protein and PEG moieties. SAXS data gave a constant radius of gyration (R(g) = 21.5 Å) for the conjugate at different concentrations and provided no evidence for an alteration of homodimeric structure or hGal-2 ellipsoidal shape upon PEGylation. In contrast, SANS data revealed a concentration dependence of R(g) for the conjugate, with the value decreasing from 31.5 Å at 2 mg/mL to 26 Å at 14 mg/mL (based on hGal-2 concentration). Scattering data have been successfully described by the model of the ellipsoidal homogeneous core (hGal-2) attached with polymer chains (PEG) at the surface. Evidently, the PEG conformation of the conjugate strongly depends on conjugate concentration and PEG's radius of gyration decreases from 24.5 to 15 Å. An excluded volume effect, arising from steric clashes between PEG molecules at high concentration, was quantified by estimating the second virial coefficient, A(2), of PEGylated hGal-2 from the SANS data. A positive value of A(2) (6.0 ± 0.4 × 10(-4) cm(3) mol g(-2)) indicates repulsive interactions between molecules, which are expected to protect the PEGylated protein against aggregation.

Molecular Insight into the Protein–Polymer Interactions in N-Terminal PEGylated Bovine Serum Albumin

“…They demonstrated that the shroud-like conformation is favored when the PEG molecular weight is higher than 10 kDa. Several other studies have been carried out to understand which configuration of PEG is favored when conjugated to proteins, − yet a definite molecular picture has not clearly emerged.…”

Conformation of the Poly(ethylene Glycol) Chains in DiPEGylated Hemoglobin Specifically Probed by SANS: Correlation with PEG Length and in Vivo Efficiency

“…In particular, based on these data, we propose a conformation of diPEG/Hb conjugates depending on two possible models, the "dumbbell" or "shroud" ones, with the latter corresponding to attached PEG chains wrapping around the protein and generating a "shielding" effect. 16,17 ■ EXPERIMENTAL SECTION Human Hemoglobin Preparation. Hb (approximately 64 000 g· mol −1 ) is a globular tetrameric protein constituted by two α and two β subunits.…”

Polymer Structure and Conformation Alter the Antigenicity of Virus-like Particle–Polymer Conjugates

“…Few studies have measured the conformation of PEGylated bioconjugates. Previous reports of the conformation of mPEG chains conjugated to proteins showed that the protein and polymer behave as two independent domains with little interaction. , …”

“…Previous reports of the conformation of mPEG chains conjugated to proteins showed that the protein and polymer behave as two independent domains with little interaction. 3,4 Recent data indicate that advanced polymer architectures provide significant advantages in improving protein efficacy in vitro and in vivo. 5 Next-generation polymers for bioconjugation are often synthesized via controlled radical polymerization, typically yielding polymers with brush-type architectures with a variety of potential side chains.…”