The limited chemical diversity of proteins constrains molecular recognition, especially in enzyme active sites. Noncanonical amino acids and efficient conjugation reactions offer powerful strategies for expanding the capabilities of protein-based therapeutics. Here, we describe the preparation and characterization of diverse conjugates on the yeast surface using systematic evaluation of fibronectin-based protein-small molecule “hybrid” conjugates. Starting from recently described fibronectin-maleimide-sulfonamide conjugates, we systematically varied the amino acid, small molecule warhead, and fibronectin clones used to generate hybrids. Following construction of a combinatorial set of hybrids, we used flow cytometric analyses to identify examples of drastic “clickable” amino acid and small molecule warhead alterations that retained isoform-specific carbonic anhydrase binding with double-digit nanomolar affinities. Interestingly, the same combination of noncanonical amino acid(s) and warhead resulted in functional conjugates when starting from 3 distinct fibronectin clones. However, no other combination of amino acid and small molecule warhead resulted in detectable levels of carbonic anhydrase binding under the conditions examined. These findings highlight promising opportunities for discovering protein-small molecule hybrids on the yeast surface by integrating a broad chemical toolkit with powerful yeast display screening and analysis platforms.

Systematic evaluation of conjugates on the yeast surface

ACS Spring 2022

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