Rapid Mapping of Protein Functional Epitopes by Combinatorial Alanine Scanning

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2000 Jul 26

PMID 10908667

Citations 141

Authors

G A Weiss

C K Watanabe

A Zhong

A Goddard

S S Sidhu

Affiliations

Soon will be listed here.

Abstract

A combinatorial alanine-scanning strategy was used to determine simultaneously the functional contributions of 19 side chains buried at the interface between human growth hormone and the extracellular domain of its receptor. A phage-displayed protein library was constructed in which the 19 side chains were preferentially allowed to vary only as the wild type or alanine. The library pool was subjected to binding selections to isolate functional clones, and DNA sequencing was used to determine the alanine/wild-type ratio at each varied position. This ratio was used to calculate the effect of each alanine substitution as a change in free energy relative to that of wild type. Only seven side chains contribute significantly to the binding interaction, and these conserved residues form a compact cluster in the human growth hormone tertiary structure. The results were in excellent agreement with free energy data previously determined by conventional alanine-scanning mutagenesis and suggest that this technology should be useful for analyzing functional epitopes in proteins.

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