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.

Keywords: Biologics; NMR Spectroscopy; PEGylated Proteins; Protein Engineering; Protein-Polymer Conjugate.

Figure 1.

Preparation of PEGylated Gal3C[T243C] and comparison of the function and temperature-dependent unfolding of Gal3C[T243C] and Gal3C[T243C]-PEG. (A) Grafting-to conjugation scheme for PEGylation of Gal3C[T243C] using a thiol-Michael addition. (B) Equilibrium binding of LacNac to Gal3C[T243C] (black) and Gal3C[T243C]-PEG (red) measured by intrinsic tryptophan fluorescence. The plotted datasets are representative single experiments, and the reported K_d values are the average K_d ± standard deviation for each protein determined from replicated experiments. (C) Thermal unfolding of Gal3C[T243C] (black) and Gal3C[T243C]-PEG (red) monitored by CD at 220 nm, showing a single cooperative unfolding event and two separate unfolding events, respectively.

Figure 2.

NMR observations of the Gal3C[T243C] structure upon PEGylation. (A) Superposition of [¹⁵N,¹H]-HSQC spectra of [u-¹⁵N]-Gal3C[T243C] (blue) and [u-¹⁵N]-Gal3C[T243C]-PEG (orange) measured at 30 °C. (B) Expanded views from (A) selected to show specific amide signals where significant changes in chemical shift or line widths were observed. (C) Surface representation of the crystal structure of Gal3C (PDB ID 4R9C). The red sphere indicates the site of chemical conjugation with PEG. Specific amino acids that showed significant changes in NMR signals are shown in stick representation and colored according to the observed difference: chemical shift perturbations (CSP) larger than 10 Hz (blue), line widths broadened by more than 25% (orange) or both chemical shift perturbations and line broadening (green). (D) Histogram of the chemical shift perturbations (blue bars) and line broadening (orange bars) observed for Gal3C[T243C]-PEG relative to the unconjugated protein as a function of the residue position. The ‘*’ indicates residues with ambiguous assignments. The green ‘#’ indicates the L114 backbone amide was broadened beyond detection for Gal3C[T243C]-PEG but not for Gal3C[T243C]. Consecutive residues that displayed significant NMR signal perturbations are highlighted by the grey boxes and are mapped on the structure in (C)

Figure 3.

Comparison of rotational diffusion rates of Gal3C[T243C] and Gal3C[T243C]-PEG. (A) Rotational correlation times (τ_c) of Gal3C[T243C] (blue) and Gal3C[T243C]-PEG (orange) at 30 °C determined from ¹⁵N relaxation data shown in (B). Error bars indicate 95% confidence intervals. (B) Global ¹⁵N longitudinal and transverse relaxation times (T₁ and T₂, respectively) of amide groups in the structured core of Gal3C[T243C] and Gal3C[T243C]-PEG. Blue points represent normalized integral values obtained for each relaxation delay, and the solid blue line is the non-linear regression used to quantify T₁ and T₂. Dotted lines indicate 95% confidence intervals from the regression procedure.

Figure 4.

Mapping Gal3C[T243C]-PEG solvent accessibility and NMR characterization of thermally cycled Gal3C[T243C]-PEG. (A) Solvent accessibility of Gal3C[T243C]-PEG amide groups determined from HDX experiments mapped onto the crystal structure of Gal3C (PDB ID 4R9C). Amides moderately or highly protected from solvent exchange over a long time scale at 30 °C are colored in pink or purple, respectively, and represented as sticks. The red sphere indicates the site of chemical conjugation with PEG. Amino acids shown in a grey dotted space-filling representation indicate residues for which significant changes were observed upon conjugation with PEG in HSQC spectra measured at 30 ºC. (B) Perturbations to amide groups after heating Gal3C[T243C]-PEG to the first transition temperature of 62 ºC and then cooling back down to 30 ºC. Data were measured at 30 ºC and compared to a freshly prepared sample of Gal3C[T243C]-PEG measured at the same temperature. Amide signals that showed chemical shift perturbations and/or line broadening are colored orange and represented as sticks. Other presentation details are the same as (A). (C) Histogram of the chemical shift perturbations (blue bars) and line broadening (orange bars) observed at 30 °C for Gal3C[T243C]-PEG after heating to 62 ºC relative to the native protein conjugate. The * indicate residues with ambiguous assignments. The green ‘#’ indicates amide signals showing very large perturbations. Consecutive residues shown in grey dotted space-filling representation in (A) and (B) are highlighted by the grey boxes.

Figure 5.

Impact of lysine residues on the spatial localization of PEG and corresponding changes to the thermal unfolding profile of PEGylated Gal3C. (A-C) Ribbon representation of Gal3C. Green colors indicate significant chemical shift perturbations or line broadening for backbone amide signals for (A) Gal3C[T243C]-PEG, (B) Gal3C[T243C,K139I]-PEG, and (C) Gal3C[T243C,K139I,K196I]-PEG upon PEGylation of the proteins. The red sphere indicates the site of chemical conjugation with PEG. For panels (B) and (C), lysines shown in black stick representation have been replaced by isoleucine in the corresponding PEGylated variant proteins. (D-I) Thermal unfolding of Gal3C and PEGylated Gal3C variants monitored by CD. Superposition of normalized CD vs. wavelength plots of measured from 30°C to 90°C for (D) Gal3C[T243C,K139I] and (E) Gal3C[T243C,K139I]-PEG. (F) Thermal unfolding of Gal3C[T243C,K139I] (black squares) and Gal3C[T243C,K139I]-PEG (red circles) monitored by CD at 224 nm. (G and H) Superposition of normalized CD vs. wavelength plots measured from 30 °C to 90 °C of (G) Gal3C[T243C,K139I,K196I] and (H) Gal3C[T243C,K139I,K196I]-PEG. (F) Thermal unfolding of Gal3C[T243C,K139I,K196I] (black squares) and Gal3C[T243C,K139I,K196I]-PEG (red circles) monitored by CD at 224 nm.