Accession Number:

AD1099213

Title:

Sequence Tolerance of a Single-Domain Antibody with a High Thermal Stability: Comparison of Computational and Experimental Fitness Profiles

Descriptive Note:

Journal Article - Embargoed Full-Text

Corporate Author:

NAVAL RESEARCH LAB WASHINGTON DC WASHINGTON United States

Report Date:

2019-06-17

Pagination or Media Count:

11.0

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.

Subject Categories:

  • Atomic and Molecular Physics and Spectroscopy
  • Computer Hardware
  • Cybernetics

Distribution Statement:

APPROVED FOR PUBLIC RELEASE