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 Mutagenesi
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- 作者：Bryan Bernat ; Miao Sun ; Mary Dwyer ; Michael Feldkamp ; and Anthony A. Kossiakoff
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：May 25, 2004
- 年：2004
- 卷：43
- 期：20
- 页码：6076 - 6084
- 全文大小：270K
- 年卷期：v.43,no.20(May 25, 2004)
- ISSN：1520-4995
文摘
Phage display mutagenesis is a widely used approach to engineering novel protein propertiesand 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 conformationaldiversity 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 affinitymatured. The modular aspect of how the mutations were grouped in the phage display libraries andcombined allowed for a systematic probing of the inherent functional cross-talk between the differentsecondary structure elements that make up the remodeled hGHv binding surface. We performed an alaninescanning analyses of 35 hGHv residues and determined the kinetics of each variant by surface plasmonresonance (SPR). This analysis showed that there is a significant difference between the additive andcooperative binding forces existing among the selected residues in each library module, and the bindingadvantage of these residues is maximized over the original wild-type residue when in the context of theother mutations in the library. The degree to which residues in a particular mutagenesis library displaybinding cooperativity characteristics is generally correlated with the conformational plasticity of thepolypeptide chain. Additionally, these cooperativity effects change when the mutations from one libraryare combined with the mutations from one or several of the other separate libraries. This supports theidea that significant functional cross-talk exists between the combined library modules that can affect thebinding energetics of individual residues over a large distance.