Selection of Specific Protein Binders for Pre-Defined Targets from an Optimized Library of Artificial Helicoidal Repeat Proteins (alphaRep)
Open Access
- 27 August 2013
- journal article
- research article
- Published by Public Library of Science (PLoS) in PLOS ONE
- Vol. 8 (8), e71512
- https://doi.org/10.1371/journal.pone.0071512
Abstract
We previously designed a new family of artificial proteins named αRep based on a subgroup of thermostable helicoidal HEAT-like repeats. We have now assembled a large optimized αRep library. In this library, the side chains at each variable position are not fully randomized but instead encoded by a distribution of codons based on the natural frequency of side chains of the natural repeats family. The library construction is based on a polymerization of micro-genes and therefore results in a distribution of proteins with a variable number of repeats. We improved the library construction process using a “filtration” procedure to retain only fully coding modules that were recombined to recreate sequence diversity. The final library named Lib2.1 contains 1.7×109 independent clones. Here, we used phage display to select, from the previously described library or from the new library, new specific αRep proteins binding to four different non-related predefined protein targets. Specific binders were selected in each case. The results show that binders with various sizes are selected including relatively long sequences, with up to 7 repeats. ITC-measured affinities vary with Kd values ranging from micromolar to nanomolar ranges. The formation of complexes is associated with a significant thermal stabilization of the bound target protein. The crystal structures of two complexes between αRep and their cognate targets were solved and show that the new interfaces are established by the variable surfaces of the repeated modules, as well by the variable N-cap residues. These results suggest that αRep library is a new and versatile source of tight and specific binding proteins with favorable biophysical properties.Keywords
This publication has 64 references indexed in Scilit:
- Calorimetric study of a series of designed repeat proteins: Modular structure and modular foldingProtein Science, 2010
- Designed Proteins To Modulate Cellular NetworksACS Chemical Biology, 2009
- Generation of new protein functions by nonhomologous combinations and rearrangements of domains and modulesCurrent Opinion in Biotechnology, 2009
- Redesign of a protein–peptide interaction: Characterization and applicationsProtein Science, 2009
- Thermodynamics, Kinetics, and Salt dependence of Folding of YopM, a Large Leucine-rich Repeat ProteinJournal of Molecular Biology, 2008
- Ligand binding by repeat proteins: natural and designedCurrent Opinion in Structural Biology, 2008
- Conformational Heterogeneity of Karyopherinβ2 Is SegmentalStructure, 2007
- Inference of Macromolecular Assemblies from Crystalline StateJournal of Molecular Biology, 2007
- High-affinity single-domain binding proteins with a binary-code interfaceProceedings of the National Academy of Sciences of the United States of America, 2007
- Engineering novel binding proteins from nonimmunoglobulin domainsNature Biotechnology, 2005