Protein-polymer conjugates are increasingly viewed as promising avenues to producing industrial enzymes with high activity capable of withstanding potentially harsh reaction conditions, or to designing novel therapeutics with triggered release, controlled masking, or increased resistance to proteolytic degradation. Common among these applications are the desire to improve the stability of protein-polymer conjugates to unfolding by exposure to chemicals or thermal stress. Thus, assays that allow researchers to robustly and easily characterize protein-polymer conjugates by obtaining thermodynamic parameters for folding stand to play an important role in the development of improved protein-polymer conjugates. Herein, we describe two techniques, differential scanning fluorimetry and intrinsic tryptophan fluorescence, used in our laboratories to obtain thermodynamic parameters of unfolding that allow for direct comparison of protein-polymer conjugates and the myriad effects of variations in attachment site, polymer identity, and polymer length. These two experiments, which are easily amenable to parallelization, are presented as high-throughput replacements for more traditionally employed circular dichroism experiments and as complements to functional chemical stability or functional thermal stability experiments. Each assay is presented in a parallelized format that allows for rapid scaling and high-throughput analysis of protein-polymer conjugate libraries. Descriptions of the assays include a discussion of advantages and disadvantages alongside protocol details and approaches to data analysis.
Keywords: Biophysical assay; Differential scanning fluorimetry; Protein–polymer conjugate; Thermodynamic parameters; Tryptophan fluorescence.
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