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Sequence Tolerance of a Single-Domain Antibody with a High Thermal Stability: Comparison of Computational and Experimental Fitness Profiles
[Image: see text] The sequence fitness of a llama single-domain antibody with an unusually high thermal stability is explored by a combined computational and experimental study. Starting with the X-ray crystallographic structure, RosettaBackrub simulations were applied to model sequence–structure to...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2019
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6648363/ https://www.ncbi.nlm.nih.gov/pubmed/31460140 http://dx.doi.org/10.1021/acsomega.9b00730 |
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author | Olson, Mark A. Legler, Patricia M. Zabetakis, Daniel Turner, Kendrick B. Anderson, George P. Goldman, Ellen R. |
author_facet | Olson, Mark A. Legler, Patricia M. Zabetakis, Daniel Turner, Kendrick B. Anderson, George P. Goldman, Ellen R. |
author_sort | Olson, Mark A. |
collection | PubMed |
description | [Image: see text] The sequence fitness of a llama single-domain antibody with an unusually high thermal stability is explored by a combined computational and experimental study. Starting with the X-ray crystallographic structure, RosettaBackrub simulations were applied to model sequence–structure tolerance profiles and identify key substitution sites. From the model calculations, an experimental site-directed mutagenesis was used to produce a panel of mutants, and their melting temperatures were determined by thermal denaturation. The results reveal a sequence fitness of an excess stability of approximately 12 °C, a value taken from a decrease in the melting temperature of an electrostatic charge-reversal substitution in the CRD3 without a deleterious effect on the binding affinity to the antigen. The tolerance for the disruption of antigen recognition without loss in the thermal stability was demonstrated by the introduction of a proline in place of a tyrosine in the CDR2, producing a mutant that eliminated binding. To further assist the sequence design and the selection of engineered single-domain antibodies, an assessment of different computational strategies is provided of their accuracy in the detection of substitution “hot spots” in the sequence tolerance landscape. |
format | Online Article Text |
id | pubmed-6648363 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-66483632019-08-27 Sequence Tolerance of a Single-Domain Antibody with a High Thermal Stability: Comparison of Computational and Experimental Fitness Profiles Olson, Mark A. Legler, Patricia M. Zabetakis, Daniel Turner, Kendrick B. Anderson, George P. Goldman, Ellen R. ACS Omega [Image: see text] The sequence fitness of a llama single-domain antibody with an unusually high thermal stability is explored by a combined computational and experimental study. Starting with the X-ray crystallographic structure, RosettaBackrub simulations were applied to model sequence–structure tolerance profiles and identify key substitution sites. From the model calculations, an experimental site-directed mutagenesis was used to produce a panel of mutants, and their melting temperatures were determined by thermal denaturation. The results reveal a sequence fitness of an excess stability of approximately 12 °C, a value taken from a decrease in the melting temperature of an electrostatic charge-reversal substitution in the CRD3 without a deleterious effect on the binding affinity to the antigen. The tolerance for the disruption of antigen recognition without loss in the thermal stability was demonstrated by the introduction of a proline in place of a tyrosine in the CDR2, producing a mutant that eliminated binding. To further assist the sequence design and the selection of engineered single-domain antibodies, an assessment of different computational strategies is provided of their accuracy in the detection of substitution “hot spots” in the sequence tolerance landscape. American Chemical Society 2019-06-17 /pmc/articles/PMC6648363/ /pubmed/31460140 http://dx.doi.org/10.1021/acsomega.9b00730 Text en Copyright © 2019 U.S. Government This is an open access article published under an ACS AuthorChoice License (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) , which permits copying and redistribution of the article or any adaptations for non-commercial purposes. |
spellingShingle | Olson, Mark A. Legler, Patricia M. Zabetakis, Daniel Turner, Kendrick B. Anderson, George P. Goldman, Ellen R. Sequence Tolerance of a Single-Domain Antibody with a High Thermal Stability: Comparison of Computational and Experimental Fitness Profiles |
title | Sequence Tolerance of a Single-Domain Antibody with
a High Thermal Stability: Comparison of Computational and Experimental
Fitness Profiles |
title_full | Sequence Tolerance of a Single-Domain Antibody with
a High Thermal Stability: Comparison of Computational and Experimental
Fitness Profiles |
title_fullStr | Sequence Tolerance of a Single-Domain Antibody with
a High Thermal Stability: Comparison of Computational and Experimental
Fitness Profiles |
title_full_unstemmed | Sequence Tolerance of a Single-Domain Antibody with
a High Thermal Stability: Comparison of Computational and Experimental
Fitness Profiles |
title_short | Sequence Tolerance of a Single-Domain Antibody with
a High Thermal Stability: Comparison of Computational and Experimental
Fitness Profiles |
title_sort | sequence tolerance of a single-domain antibody with
a high thermal stability: comparison of computational and experimental
fitness profiles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6648363/ https://www.ncbi.nlm.nih.gov/pubmed/31460140 http://dx.doi.org/10.1021/acsomega.9b00730 |
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