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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 |
Sumario: | [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. |
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