TY - JOUR
T1 - Determinants of Developability and Evolvability of Synthetic Miniproteins as Ligand Scaffolds
AU - McConnell, Adam
AU - Batten, Sun Li
AU - Hackel, Benjamin J.
N1 - Publisher Copyright:
© 2023 Elsevier Ltd
PY - 2023/12/15
Y1 - 2023/12/15
N2 - Binding ligands empower molecular therapeutics and diagnostics. Despite an array of protein scaffolds engineered for binding, the biophysical elements that drive developability and evolvability are not fully understood. In particular, engineering novel function while maintaining biophysical integrity within the context of small, single-domain proteins is challenged by integration of the structural framework and the evolved binding site. Miniproteins present a challenge to our limits of protein engineering capability and provide advantages in physiological targeting, modularity for multi-functional constructs, and unique binding modes. Herein, we evaluate the ability of hyperstable synthetic miniproteins, originally designed for foldedness, to function as binding scaffolds. We synthesized 45 combinatorial libraries, with 109 variants, systematically varied across two topologies, each with five starting frameworks and four or five diverse, structurally distinct paratopes, to elucidate their impact on evolvability and developability. We evaluated evolvability with yeast display binding selections against four targets. High-throughput assays –stability via yeast display and soluble expression via split-GFP in E. coli – measured developability. The comprehensive, robust dataset demonstrates how protein topology, parental framework, and paratope structure and location all impact scaffold performance. A hyperstable framework and localized diversity are not sufficient for an effective scaffold, but several designs of these elements within synthetic miniproteins designed solely for stability result in scaffold libraries with effective evolvability and developability. Engineered variants were well-folded, thermally stable, and bound target with single-digit nanomolar affinity. Thus, hyperstable synthetic miniproteins can serve as precursors to developable, evolvable mini-scaffolds with unique potential for physiological transport, modularity, and binding modes.
AB - Binding ligands empower molecular therapeutics and diagnostics. Despite an array of protein scaffolds engineered for binding, the biophysical elements that drive developability and evolvability are not fully understood. In particular, engineering novel function while maintaining biophysical integrity within the context of small, single-domain proteins is challenged by integration of the structural framework and the evolved binding site. Miniproteins present a challenge to our limits of protein engineering capability and provide advantages in physiological targeting, modularity for multi-functional constructs, and unique binding modes. Herein, we evaluate the ability of hyperstable synthetic miniproteins, originally designed for foldedness, to function as binding scaffolds. We synthesized 45 combinatorial libraries, with 109 variants, systematically varied across two topologies, each with five starting frameworks and four or five diverse, structurally distinct paratopes, to elucidate their impact on evolvability and developability. We evaluated evolvability with yeast display binding selections against four targets. High-throughput assays –stability via yeast display and soluble expression via split-GFP in E. coli – measured developability. The comprehensive, robust dataset demonstrates how protein topology, parental framework, and paratope structure and location all impact scaffold performance. A hyperstable framework and localized diversity are not sufficient for an effective scaffold, but several designs of these elements within synthetic miniproteins designed solely for stability result in scaffold libraries with effective evolvability and developability. Engineered variants were well-folded, thermally stable, and bound target with single-digit nanomolar affinity. Thus, hyperstable synthetic miniproteins can serve as precursors to developable, evolvable mini-scaffolds with unique potential for physiological transport, modularity, and binding modes.
KW - biophysical robustness
KW - evolvability
KW - molecular targeting
KW - protein engineering
KW - topology
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U2 - 10.1016/j.jmb.2023.168339
DO - 10.1016/j.jmb.2023.168339
M3 - Article
C2 - 37923119
AN - SCOPUS:85176497574
SN - 0022-2836
VL - 435
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 24
M1 - 168339
ER -