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Protein-polymer conjugates are a blooming class of hybrid systems with high biomedical potential. Despite a plethora of papers on their biomedical properties, the physical-chemical characterization of many protein-polymer conjugates is missing. Here, we evaluated the thermal stability of a set of fully-degradable polyphosphoester-protein conjugates by variable temperature circular dichroism, a common but powerful technique. We extensively describe their thermodynamic stability in different environments (in physiological buffer or in presence of chemical denaturants, e.g., acid or urea), highlighting the protective role of the polymer in preserving the protein from denaturation. For the first time, we propose a simple but effective protocol to achieve useful information on these systems in vitro, useful to screen new samples in their early stages., (© 2022 The Authors. Chirality published by Wiley Periodicals LLC.)

Polyphosphoesters (PPEs), a versatile class of biodegradable and biocompatible polymers, have been proposed as alternatives to poly(ethylene glycol) (PEG), which is suspected to be responsible for anaphylactic reactions in some patients after the administration of PEGylated compounds, e.g., in the current Covid-19 vaccines. We present the synthesis and characterization of a novel set of protein-polymer conjugates using the model protein myoglobin and a set of PPEs with different hydrophilicity and molar mass. We report an extensive evaluation of the (bio)physical properties of the protein within the conjugates, studying its conformation, residual activity, and thermal stability by complementary techniques (UV-vis spectroscopy, nano-differential scanning calorimetry, and fluorometry). The data underline the systematic influence of polymer hydrophilicity on protein properties. The more hydrophobic polymers destabilize the protein, the more hydrophilic PPEs protect against thermally induced aggregation and proteolytic degradation. This basic study aims at guiding the design of future PPEylated drugs and protein conjugates.

The bio and chemical physics of protein-polymer conjugates are related to parameters that characterize each component. With this work, it is intended to feature the dynamical properties of the protein-polymer conjugate myoglobin (Mb)-poly(ethyl ethylene phosphate), in the ps and ns time scales, in order to understand the respective roles of the protein and of the polymer size in the dynamics of the conjugate. Elastic and quasi-elastic neutron scattering is performed on completely hydrogenated samples with variable number of polymer chains covalently attached to the protein. The role of the polymer length in the protein solvation and internal dynamics is investigated using two conjugates formed by polymers of different molecular weight. It is confirmed that the flexibility of the complex increases with the number of grafted polymer chains and that a sharp dynamical transition appears when either grafting density or polymer molecular weight are high. It is shown that protein size is crucial for the polymer structural organization and interaction on the protein surface and it is established that the glass properties of the polymer change upon conjugation. The results give a better insight of the equivalence of the polymer coating and the role of water on the surface of proteins., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Graphical abstract Highlights • The myoglobin denaturation mechanism remains unaffected upon PEGylation. • PEGylation causes a thermal destabilization of myoglobin but enhances reversibility. • PEGylation decreases the solvent-exposed surface of the myoglobin denatured state. Abstract PEGylated proteins are widely used for therapeutic applications, therefore a fundamental understanding of the conjugates' structure and their behaviour in solution is essential to promote new developments in this field. In the present work, myoglobin-poly(ethylene glycol) conjugates were synthesized and studied by differential scanning calorimetry and UV–vis spectroscopy to obtain information on the bioconjugates' thermodynamic stability, also focusing on PEG's role on the solvent-protein surface interaction. The overall results of this study indicated a thermal destabilization of the protein that follows the extent of the bioconjugation without, however, compromising the native structure which remains functional. Moreover, the myoglobin PEGylation prevented the post-denaturation aggregation phenomena and enhanced the protein thermal reversibility. The thermodynamic interpretation of the data indicated that the bioconjugation influences the solvent-exposed protein surface difference between native and denatured state, contributing to the interpretation of the overall protein modification and functionality. [ABSTRACT FROM AUTHOR]