Polymer-based protein engineering (PBPE) by surface-initiated atom transfer radical polymerization (ATRP) induces structural changes to the protein by generating protein-polymer conjugates with dense polymer shells that impact protein function. Protein-polymer conjugate properties are considerably influenced by the site of modification on the protein surface combined with the chemical and physical properties of the polymer. In this presentation, we have focused on determining the site and rate of reaction between amine-reactive ATRP initiators and protein amino groups as a step forward in controlling protein-initiator reactions. We performed a systematic characterization of the reaction between a bromine-functionalized N-hydroxysuccinimide amine-reactive ATRP initiator and the amino groups in two model proteins, lysozyme and chymotrypsin. The tertiary structures of the proteins were used to predict computationally α-amino group and lysine side-chain exposed surface area (ESA) and analyze the chemical and structural environment of the amino groups. A probe radius resembling the size of the initiator was used in the ESA calculations to accurately model amino group accessibility to ATRP initiator molecules. Enzymatic and chemical digestion studies of protein-initiator complexes followed by peptide mass mapping were used to identify initiator modification sites. Experimental data showed that the rate of initiator-protein modification at each amine site was primarily dependent on surface accessibility. Further refinements of the prediction of where the initiator modified the protein, and in what sequence, were achieved by considering p<i>K</i><sub>a</sub>, steric hindrance, local charge and environment of each amino group. These factors were combined to generate a rules-based decision tree that could be used to predict the site and relative reactivity of the amino groups on any protein when modified with ATRP initiators. We are now using the decision tree to predictably modify other proteins for polymer-based protein engineering.

Tertiary structure-based prediction of how ATRP initiators react with proteins

254th National Meeting (2017)

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