Atomic‐Scale View of Protein‐PEG Interactions that Redirect the Thermal Unfolding Pathway of PEGylated Human Galectin‐3

Abstract

PEGylation is a promising approach to address the central challenge of applying biologics, i.e., lack of protein stability in the demanding environment of the human body. Wider application is hindered by lack of atomic level understanding of protein-PEG interactions, preventing design of conjugates with predicted properties. We deployed an integrative structural and biophysical approach to address this critical challenge with the PEGylated carbohydrate recognition domain of human galectin-3 (Gal3C), a lectin essential for cell adhesion and potential biologic. PEGylation dramatically increased Gal3C thermal stability, forming a stable intermediate and redirecting its unfolding pathway. Structural details revealed by NMR pointed to a potential role of PEG localization facilitated by charged residues. Replacing these residues subtly altered the protein-PEG interface and thermal unfolding behavior, providing insight into rationally designing conjugates while preserving PEGylation benefits.

Conserved Protein–Polymer Interactions across Structurally Diverse Polymers Underlie Alterations to Protein Thermal Unfolding

“…The impact of conjugation of Gal3C with PDMA 61 was further visualized by mapping residues showing either significant chemical shift perturbations (>10 Hz) or line broadening onto a structure of Gal3C (PDB ID 4R9A) 40 (Figure 3). The largest impact was observed for residues local to the site of chemical conjugation, similar to Gal3C conjugates prepared with PEG, 33 and similar patterns of effects on the Gal3C backbone were observed for PEG and PDMA 61 (Figure 3). Though monomers for PDMA 61 and PEG have different chemical structures (Figure 1), both polymers have linear architecture, indicating that similar polymer architectures result in similar patterns of protein−polymer interactions (Figure 3).…”

“…Samples used for biophysical analyses were therefore prepared under nonreducing conditions. Minor heterogeneity in linker chemistry was not concerning based on previous work which suggests the linker has little effect on the galectin structure 33 or function. 37 HSQC spectra of Gal3C[T243C] conjugates did not show the presence of extensive peak doubling, as would be expected for heterogeneous samples containing a significant fraction of unconjugated protein (see below).…”

“…The polymers were conjugated to Gal3C­[T243C] with a grafting-to scheme (Figure A). An excess of each aqueous polymer stock was used, with 10 equiv of reactive polymer-maleimide present to participate in a thiol-Michael addition with the solvent-exposed cysteine, C243, over the course of 4 h. Previously, it was determined that only C243 was functionalized with 5400 g/mol PEG-maleimide under similar grafting-to conditions with a shorter reaction time, and thus we anticipated site specificity would be preserved …”

“…Gal3C[T243C] was expressed and purified according to earlier reports. 33 Gal3C[T243C] conjugates were generated with a grafting-to method using thiol-Michael addition between the polymer-maleimide and the protein at position C243. Unreacted Gal3C[T243C] and excess polymer were removed using lactose-affinity chromatography followed by size exclusion chromatography.…”

How to Characterize the Protein Structure and Polymer Conformation in Protein‐Polymer Conjugates – a Perspective

“…Harris, Eddy, and coworkers recently demonstrated that PEG interacts with the protein surface of human galectin‐3C. [ 57 ] Site‐specific PEGylation was performed after the biotechnological incorporation of cysteine into the protein. The polymer has a negligible impact on the protein structure but interacts with the protein surface in a specific region.…”