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Accession Number:
AD1099213
Title:
Sequence Tolerance of a Single-Domain Antibody with a High Thermal Stability: Comparison of Computational and Experimental Fitness Profiles
Corporate Author:
NAVAL RESEARCH LAB WASHINGTON DC WASHINGTON United States
Report Date:
2019-06-17
Abstract:
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 sequencestructure 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 deg. 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.
Descriptive Note:
Journal Article - Embargoed Full-Text
Supplementary Note:
Acs Omega , 4, 6, 01 Jan 0001, 01 Jan 0001,
Pages:
0011
Distribution Statement:
Approved For Public Release;
File Size:
3.93MB
