Intramolecular Interactions of Conjugated Polymers Mimic Molecular Chaperones to Stabilize Protein–Polymer Conjugates

Abstract

The power and elegance of protein-polymer conjugates has solved many vexing problems for society. Rational design of these complex covalent hybrids depends on a deep understanding of how polymer physicochemical properties impact the conjugate structure-function-dynamic relationships. We have generated a large family of chymotrypsin-polymer conjugates which differ in polymer length and charge, using grafting-from atom-transfer radical polymerization, to elucidate how the polymers influenced enzyme structure and function at pHs that would unfold and inactivate the enzyme. We also used molecular dynamics simulations to deepen our understanding of protein-polymer intramolecular interactions. Remarkably, the data revealed that, contrary to current thoughts on how polymers stabilize proteins, appropriately designed polymers actually stabilize partially unfolded intermediates and assist in refolding to an active conformation. Long, hydrophilic polymers minimized interfacial interactions in partially unfolded conjugates leading to increased stabilization. The design of covalently attached intramolecular biomimetic chaperones that drive protein refolding could have far reaching consequences.

Molecular Simulations of PEGylated Biomolecules, Liposomes, and Nanoparticles for Drug Delivery Applications

“…Using this CG model, they simulated PEG interacting with plasma proteins such as bovine serum albumin, human serum albumin, and apo-human serum transferrin, which reasonably predict the experimentally observed local densities of PEG around individual amino acids [65,66]. In particular, they simulated PEGylated chymotrypsin (a digestive enzyme), showing that PEG chains stabilize partially unfolded intermediates and even help the refolding to an active conformation, to an extent dependent on pH as described in Figure 2 [67], which supports the experimental hypothesis regarding the effect of PEG on protein folding and helps in the rational design of protein-polymer conjugates. They also observed the dependence of the PEG-peptide hydrogel interaction on peptide sequence and solvent condition [68].…”

“…To overcome this, PEGylation has been experimentally applied to AMPs such as nisin [72], magainin 2, tachyplesin I [73,74], KYE 2 8 [75], LL-37 [76] and synthetic AMPs (CaLL [77] and M33 [78]), showing decreased antimicrobial activity and increased solubility, which has motivated simulation studies on the interactions between AMP and PEG. [67]. Copyright (2018) American Chemical Society).…”

“… Unfolding pathways at different pH for ( a ) native chymotrypsin at pH 1 and 12, ( b ) PEGylated chymotrypsin at pH 1, and ( c ) PEGylated chymotrypsin at pH 12. PEG chains stabilize partially unfolded intermediate states and thus inhibit irreversible denaturation at pH 1 but not at pH 12 (reprinted with permission from [ 67 ]. Copyright (2018) American Chemical Society).…”

Tunable Polymeric Scaffolds for Enzyme Immobilization

“…Indeed, several works evaluating the effect of these parameters on the bioactivity have been published in the last years ( Table 1 ). Thus, Baker et al (2018) synthetized different chymotrypsin (α-CT)-polymer conjugates varying polymer charge, hydrophobicity, and molecular weight. They conjugated zwitterionic PCBMA, neutral POEGMA, neutral to positive poly(dimethylamino)ethyl methacrylate (PDMAEMA), positive quaternary ammonium ion-containing polymers (PQA), and negative poly(styrene–maleic anhydride) (PSMA) of three different chain lengths each.…”

Mapping protein–polymer conformations in bioconjugates with atomic precision

“…There is no consensus on the impact of polymer–protein non-covalent associations and conformation on stability. 16 , 17 , 27 , 28 One model proposes that protein–polymer associations are stabilizing through non-covalent bonds, 17 others suggest protein–polymer associations are destabilizing by disrupting the hydration shell, 28 or that the polymer acts as a chaperone. 16 The dearth of experimental data and conflicting models limit the mechanistic understanding of protein–polymer hybrids and rational bioconjugate engineering.…”