The study of protein stability and bioactivity within a confined space has important ramifications across several areas ranging from protein therapeutics to food industry. The long-term stability and targeted delivery of proteins is still an elusive goal. Proteins have been usually studied in diluted solution or as single protein not allowing to understand the important effect of protein-protein interaction. This lead to study the effect of volume exclusion by one soluble macromolecule on another ('macromolecular crowding'), as well as assessing the effect of volume exclusion on a soluble macromolecule ('macromolecular confinement').While in the last few years several studies provide interesting insights regarding protein dynamics, they are often limited by the strong interaction between the confinement/crowding agent and the protein. In the present work we report the use of polymersomes formed by amphiphilic block copolymers PMPC25-PDPA70 in PBS to encapsulate myoglobin under conditions close to those encountered within living cells. We show that confinement within polymersomes leads to a significant improvement in protein stability against thermal denaturation up to 950C at neutral pH. Circular dichroism, fluorescence and UV.VIS. were used to study the protein structure. The results showed little or no evidence of unfolding or reduced enzymatic activity. The latter parameter actually exhibits a two-fold increase after thermal cycling when the confined protein concentration is higher than 5% v/v. Our results suggest that nanoscopic confinement is a promising new route for the enhanced long-term storage of proteins. In conclusion, we provide strong evidence that protein thermodynamics within a nanoscopic aqueous environment are strongly affected by both concentration and confinement. More importantly, we demonstrate that proteins encapsulated within polymersomes can withstand large temperature gradients without compromising their structure (and hence their biochemical activity). This is very important in the context of polymersome-mediated delivery of proteins and/or the development of nano-reactors.

Protein behaviour in biomimetic nanoscopic environment

248th National Meeting (2014)

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