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Thermal stability of single-domain antibodies estimated by molecular dynamics simulations.

ABSTRACT: Single-domain antibodies (sdAbs) function like regular antibodies, however, consist of only one domain. Because of their low molecular weight, sdAbs have advantages with respect to production and delivery to their targets and for applications such as antibody drugs and biosensors. Thus, sdAbs with high thermal stability are required. In this work, we chose seven sdAbs, which have a wide range of melting temperature (Tm ) values and known structures. We applied molecular dynamics (MD) simulations to estimate their relative stability and compared them with the experimental data. High-temperature MD simulations at 400 K and 500 K were executed with simulations at 300 K as a control. The fraction of native atomic contacts, Q, measured for the 400 K simulations showed a fairly good correlation with the Tm values. Interestingly, when the residues were classified by their hydrophobicity and size, the Q values of hydrophilic residues exhibited an even better correlation, suggesting that stabilization is correlated with favorable interactions of hydrophilic residues. Measuring the Q value on a per-residue level enabled us to identify residues that contribute significantly to the instability and thus demonstrating how our analysis can be used in a mutant case study.

SUBMITTER: Bekker GJ

PROVIDER: S-EPMC6319760 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Thermal stability of single-domain antibodies estimated by molecular dynamics simulations.

Bekker Gert-Jan GJ Ma Benson B Kamiya Narutoshi N

Protein science : a publication of the Protein Society 20181220 2

Single-domain antibodies (sdAbs) function like regular antibodies, however, consist of only one domain. Because of their low molecular weight, sdAbs have advantages with respect to production and delivery to their targets and for applications such as antibody drugs and biosensors. Thus, sdAbs with high thermal stability are required. In this work, we chose seven sdAbs, which have a wide range of melting temperature (T<sub>m</sub> ) values and known structures. We applied molecular dynamics (MD) ...[more]

PMID: 30394618

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Project description:The extreme pH and protease-rich environment of the upper gastrointestinal tract is a major obstacle facing orally-administered protein therapeutics, including antibodies. Through protein engineering, several Clostridium difficile toxin A-specific heavy chain antibody variable domains (V(H)Hs) were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. Mutant antibodies were compared to their wild-type counterparts with respect to expression yield, non-aggregation status, affinity for toxin A, circular dichroism (CD) structural signatures, thermal stability, protease resistance, and toxin A-neutralizing capacity. The mutant V(H)Hs were found to be well expressed, although with lower yields compared to wild-type counterparts, were non-aggregating monomers, retained low nM affinity for toxin A, albeit the majority showed somewhat reduced affinity compared to wild-type counterparts, and were capable of in vitro toxin A neutralization in cell-based assays. Far-UV and near-UV CD spectroscopy consistently showed shifts in peak intensity and selective peak minima for wild-type and mutant V(H)H pairs; however, the overall CD profile remained very similar. A significant increase in the thermal unfolding midpoint temperature was observed for all mutants at both neutral and acidic pH. Digestion of the V(H)Hs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants. Mutant V(H)H trypsin resistance was similar to that of wild-type V(H)Hs, although the trypsin resistance of one V(H)H mutant was significantly reduced. Therefore, the introduction of a second disulfide bond in the hydrophobic core not only increases V(H)H thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase V(H)H stability at low pH and impart protease resistance, with only minor perturbations in target binding affinities. These are all desirable characteristics for the design of protein-based oral therapeutics.

Dataset Information

Thermal stability of single-domain antibodies estimated by molecular dynamics simulations.

Publications

Thermal stability of single-domain antibodies estimated by molecular dynamics simulations.

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