Ligand-Binding Cooperativity Effects in Polymer–Protein Conjugation

Abstract

We present an electron paramagnetic resonance (EPR) spectroscopic characterization of structural and dynamic effects that stem from post-translational modifications of bovine serum albumin (BSA), an established model system for polymer–protein conjugation. Beyond the typical drug delivery and biocompatibility aspect of such systems, we illustrate the causes that alter internal dynamics and therefore functionality in terms of ligand-binding to the BSA protein core. Uptake of the paramagnetic fatty acid derivative 16-doxyl stearic acid by several BSA-based squaric acid macroinitiators and polymer–protein conjugates was studied by EPR spectroscopy, aided by dynamic light scattering (DLS) and zeta potential measurements. The conjugates were grafted from oligo­(ethylene glycol) methyl ether methacrylate (OEGMA), forming an overall core–shell-like structure. It is found that ligand-binding and associated parameters such as binding affinity, cooperativity, and the number of binding sites of BSA change drastically with the extent of surface modification. In the course of processing BSA, the ligands also change their preference for individual binding sites, as observed from a comparative view of their spatial alignments in double electron electron resonance (DEER) experiments. The protein-attached polymers constitute a diffusion barrier that significantly hamper ligand uptake. Moreover, zeta potentials (ζ) decrease linearly with the degree of surface modification in protein macroinitiators and an effective dielectric constant can be estimated for the polymer layer in the conjugates. All this suggests that ligand uptake characteristics in BSA can be fine-tuned by the extent and nature of such post-translational modifications (PTMs). We show that EPR spectroscopy is suitable for quantifying these subtle PTM-based functional effects from self-assembly of substrate and ligand.

Fatty Acid Binding to Human Serum Albumin in Blood Serum Characterized by EPR Spectroscopy

“…As an example, the experimental and simulated spectra of all loading ratios of a healthy individual (sample numbered 1 in control group) are given in Figure (a). As we have reported earlier,,, the EPR spectra at these loading ratios of HSA:FA are comprised of three spectral fractions, as shown in Figure (b): (A) strongly bound and immobilized FAs that have high rotational correlation times ( ≥10 ns); (B) FA that are intermediately‐strong bound ( ≥1 ns); and (C) FAs that float relatively free in solution ( ≥0.1 ns): The rigorously simulated EPR spectra thus always consist of these three fractions in different weights, of course the and hyperfine coupling values were always allowed to vary during the simulation to a small degree.…”

“…As an example, the experimental and simulated spectra of all loading ratios of a healthy individual (sample numbered 1 in control group) are given in Figure 1(a). As we have reported earlier [12,13,15,16] the EPR spectra at these loading ratios of HSA:FA are comprised of three spectral fractions, as shown in Figure 1 simulated EPR spectra thus always consist of these three fractions in different weights, of course the t c and hyperfine coupling values were always allowed to vary during the simulation to a small degree.…”

“…Electron paramagnetic Resonance (EPR) is a powerful tool which can monitor such structural and therefore functional modifications . EPR spectroscopy as a magnetic resonance method detects spins of unpaired electron, hence (unlike in nuclear magnetic resonance (NMR) spectroscopy) one needs to introduce paramagnetic species into the sample.…”

“…[11] Electron paramagnetic Resonance (EPR) is a powerful tool which can monitor such structural and therefore functional modifications. [12][13][14][15][16] EPR spectroscopy as a magnetic resonance method detects spins of unpaired electron, hence (unlike in nuclear magnetic resonance (NMR) spectroscopy) one needs to introduce paramagnetic species into the sample. Here, the labeled stearic acid (16-doxyl stearic acid, 16-DSA) is our main probe molecule of choice and is used as a spin probe throughout this study.…”

Fatty Acid Triangulation in Albumins Using a Landmark Spin Label

“…Particularly, a recent study proved that EPR spectroscopy is capable to elucidate the intrinsic entanglement of such intricate structural and dynamic properties when albumin is exposed to post‐translational modifications (PTMs). Utilizing the DSA‐probed BSA model system, it could be shown how specific PTMs may exert strong alterations on albumin‐ligand interactions ( N T , K A ) that are again mirrored in corresponding ligand alignments and altered binding site occupations ( P ( r )), respectively …”