DOI: 10.1073/pnas.160252097. Rapid mapping of protein functional epitopes by combinatorial alanine scanning

Rapid mapping of protein functional epitopes by combinatorial alanine scanning

10.1073/pnas.160252097

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

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.

Bibliography

Weiss, G. A., Watanabe, C. K., Zhong, A., Goddard, A., & Sidhu, S. S. (2000). Rapid mapping of protein functional epitopes by combinatorial alanine scanning. Proceedings of the National Academy of Sciences, 97(16), 8950â8954.

Authors 5

Gregory A. Weiss (first)
Colin K. Watanabe (additional)
Alan Zhong (additional)
Audrey Goddard (additional)
Sachdev S. Sidhu (additional)

References 30 Referenced 294

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Dates

Type	When
Created	23 years, 1 month ago (July 26, 2002, 10:35 a.m.)
Deposited	3 years, 4 months ago (April 13, 2022, 12:46 a.m.)
Indexed	2 days, 8 hours ago (Aug. 27, 2025, 11:50 a.m.)
Issued	25 years, 1 month ago (July 25, 2000)
Published	25 years, 1 month ago (July 25, 2000)
Published Online	25 years, 1 month ago (July 25, 2000)
Published Print	25 years ago (Aug. 1, 2000)

Funders 0

None

BibTeX

@article{Weiss_2000, title={Rapid mapping of protein functional epitopes by combinatorial alanine scanning}, volume={97}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.160252097}, DOI={10.1073/pnas.160252097}, number={16}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Weiss, Gregory A. and Watanabe, Colin K. and Zhong, Alan and Goddard, Audrey and Sidhu, Sachdev S.}, year={2000}, month=jul, pages={8950–8954} }

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