The rugged protein sequence-function landscape complicates efforts, both in nature and in the laboratory, to evolve protein function. Protein library diversification must strike a balance between sufficient variegation to thoroughly sample alternative functionality versus the probability of mutant destabilization below an expressible threshold. In this work, we explore the sequence-function landscape in the context of screening for molecular recognition from an Ig scaffold library. The fibronectin type III domain is used to explore the impact of two sequence diversification strategies: (a) partial wild-type conservation at structurally important positions within the paratope region and (b) tailored amino acid composition mimicking antibody binding-site composition at putative paratope positions. Structurally important positions within the paratope region were identified through stability, structural, and phylogenetic analyses and partially or fully conserved in sequence. To achieve tailored antibody-like diversity, we designed a set of skewed nucleotide mixtures yielding codons approximately matching the distribution observed in antibody complementarity-determining regions without incurring the expense of triphosphoramidite-based construction. These design elements were explored via comparison of three library designs: a random library, a library with wild-type bias in the DE loop only and tyrosine-serine diversity elsewhere, and a library with wild-type bias at 11 positions and the antibody-inspired amino acid distribution. Using pooled libraries for direct competition in a single tube, selection and maturation of binders to seven targets yielded 19 of 21 clones that originated from the structurally biased, tailored-diversity library design. Sequence analysis of the selected clones supports the importance of both tailored compositional diversity and structural bias. In addition, selection of both well and poorly expressed clones from two libraries further elucidated the impact of structural bias.
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