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A Gradient of Sitewise Diversity Promotes Evolutionary Fitness for Binder Discovery in a Three-Helix Bundle Protein Scaffold
- Source :
- Biochemistry. 56(11)
- Publication Year :
- 2017
-
Abstract
- Engineered proteins provide clinically and industrially impactful molecules and utility within fundamental research. Yet, inefficiencies in discovering lead variants with new desired function, while maintaining stability, hinder progress. Improved function, which can result from a few strategic mutations, is fundamentally separate from discovering novel function, which often requires large leaps in sequence space. While a highly diverse combinatorial library covering immense sequence space would empower protein discovery, the ability to sample only a minor subset of sequence space and the typical destabilization of random mutations preclude this strategy. A balance must be reached. At library scale, compounding several destabilizing mutations renders many variants unable to properly fold and devoid of function. Broadly searching sequence space while reducing destabilization may enhance evolution. We exemplify this balance with affibody, a three-helix bundle protein scaffold. Using natural ligand datasets, stability and structural computations, and deep sequencing thousands of binding variants, a protein library was designed on a sitewise basis with a gradient of mutational levels across 29% of the protein. In direct competition of biased and uniform libraries, both with 1×109 variants, for discovery of 6×104 ligands (5×103 clusters) toward seven targets, biased amino acid frequency increased ligand discovery 13±3-fold. Evolutionarily favorable amino acids, both globally and site-specifically, are further elucidated. The sitewise amino acid bias aids evolutionary discovery by reducing mutant destabilization as evidenced by 15°C higher midpoint of denaturation relative to unbiased mutants (62±4°C vs. 47±11°C, p
- Subjects :
- 0301 basic medicine
Scaffold protein
Models, Molecular
Protein Denaturation
B7 Antigens
Computational biology
Saccharomyces cerevisiae
Biology
Glucosephosphate Dehydrogenase
Protein Engineering
Biochemistry
Protein Structure, Secondary
Article
Receptors, G-Protein-Coupled
03 medical and health sciences
Peptide Library
Humans
Helix bundle
Genetics
Protein Stability
Transferrin
Cytochromes c
Proto-Oncogene Proteins c-met
Receptors, TNF-Related Apoptosis-Inducing Ligand
030104 developmental biology
Bundle
Immunoglobulin G
Mutation
Muramidase
Sequence space (evolution)
Directed Molecular Evolution
Function (biology)
Protein Binding
Subjects
Details
- ISSN :
- 15204995
- Volume :
- 56
- Issue :
- 11
- Database :
- OpenAIRE
- Journal :
- Biochemistry
- Accession number :
- edsair.doi.dedup.....480db4baee6a64cf0efaf409b0c11a38