(78b) Gradient Diversity Enriches Combinatorial Protein Library Design

Efficient evolution of molecular recognition reagents is vital to clinical targeting and biotechnology. We examined the hypothesis that the most efficient evolution is achieved with a combinatorial library exhibiting a gradient of diversity from extensive diversity in the paratope hot spot to full conservation in the framework. Importantly, this gradient includes moderate diversity, with structural bias, within the paratope interfacing with target yet peripheral to the hot spot. Moreover, more mild diversity is included adjacent to the interfacial residues to yield optimal intramolecular contacts with the newly identified paratope. We assessed this approach to library design within the context of two distinct protein topologies: the beta sandwich type III fibronectin domain with the paratope in three loops and the three-helix bundle affibody domain with the paratope on one surface of two helices. A range of diversities was introduced into sites throughout the paratope and its periphery. The resultant naïve combinatorial libraries were screened for specific binding to four different protein targets using yeast surface display and magnetic and fluorescence binding selections. Thorough sequencing of binding ligands quantitatively revealed the evolutionary efficacy of each diversity option at each site. The proposed evolutionary benefit of gradient diversity was evident from the sequence analysis. Current work on second-generation libraries, and continued expansion of the target pool, will fine tune the optimal diversity for each site within both topologies. These results improve the evolutionary efficiency of two validated ligand scaffolds and provide a generalizable roadmap for library design in other scaffolds, which is facilitated by the current ease and power of high throughput selection and sequencing.