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


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 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.

Submission Details

ID: Py9nZcnz3

Submitter: Shu-Ching Ou

Submission Date: June 24, 2019, 8:52 p.m.

Version: 1

Publication Details
Olson MA;Legler PM;Zabetakis D;Turner KB;Anderson GP;Goldman ER,ACS Omega (2019) Sequence Tolerance of a Single-Domain Antibody with a High Thermal Stability: Comparison of Computational and Experimental Fitness Profiles
Additional Information

Study Summary

Number of data points 38
Proteins Single Domain Antibody
Unique complexes 38
Assays/Quantities/Protocols Experimental Assay: Tm ; Experimental Assay: Kd
Libraries variants for A3 ; variants for complex

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)