title: Dissecting the binding energy epitope of a high-affinity variant of human growth hormone: cooperative and additive effects from combining mutations from independently selected phage display mutagenesis libraries.
abstract: Phage display mutagenesis is a widely used approach to engineering novel protein properties and is especially powerful in probing structure-function relationships in molecular recognition processes. The relative contributions of additive and cooperative binding forces and the influence of conformational diversity in producing a novel protein-protein interface is investigated using as a model an ultra-high-affinity receptor binding variant of human growth hormone (hGHv) that has been previously affinity matured. The modular aspect of how the mutations were grouped in the phage display libraries and combined allowed for a systematic probing of the inherent functional cross-talk between the different secondary structure elements that make up the remodeled hGHv binding surface. We performed an alanine scanning analyses of 35 hGHv residues and determined the kinetics of each variant by surface plasmon resonance (SPR). This analysis showed that there is a significant difference between the additive and cooperative binding forces existing among the selected residues in each library module, and the binding advantage of these residues is maximized over the original wild-type residue when in the context of the other mutations in the library. The degree to which residues in a particular mutagenesis library display binding cooperativity characteristics is generally correlated with the conformational plasticity of the polypeptide chain. Additionally, these cooperativity effects change when the mutations from one library are combined with the mutations from one or several of the other separate libraries. This supports the idea that significant functional cross-talk exists between the combined library modules that can affect the binding energetics of individual residues over a large distance.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Cummings Life Science Center, 920 East 58 Street, Chicago, Illinois 60637, USA.
authors.forename: Bryan
authors.initials: B
authors.lastname: Bernat
authors.forename: Miao
authors.initials: M
authors.lastname: Sun
authors.forename: Mary
authors.initials: M
authors.lastname: Dwyer
authors.forename: Michael
authors.initials: M
authors.lastname: Feldkamp
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Kossiakoff
mesh_terms: D000939:Epitopes; D019382:Human Growth Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D019151:Peptide Library; D011485:Protein Binding; D017433:Protein Structure, Secondary; D020349:Surface Plasmon Resonance
chemical_list: D000939:Epitopes; D019151:Peptide Library; D019382:Human Growth Hormone