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 (T m) 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 T m 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.


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Top view Figure S12: Average structures obtained during each of the 10 parallel trajectories at 400 K for 4idl colored by the average per-residue Q-value (0 to 1: red to blue), with excluded residues in white. The CDR regions are indicated in magenta denoted as CDR1, CDR2 and CDR3 and are placed at the top of each loop. The residues involved in our mutations (Asn27, Arg71) are shown, colored in cyan and denoted in black text.

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Back view Top view Figure S13: Average structures obtained during each of the 10 parallel trajectories at 400 K for 1fvc colored by the average per-residue Q-value (0 to 1: red to blue), with excluded residues in white. The CDR regions are indicated in magenta denoted as CDR1, CDR2 and CDR3 and are placed at the top of each loop.

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Top view Figure S14: Average structures obtained during each of the 10 parallel trajectories at 400 K for 4w70 colored by the average per-residue Q-value (0 to 1: red to blue), with excluded residues in white. The CDR regions are indicated in magenta denoted as CDR1, CDR2 and CDR3 and are placed at the top of each loop.

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Back view Top view Figure S15: Average structures obtained during each of the 10 parallel trajectories at 400 K for 1mel colored by the average per-residue Q-value (0 to 1: red to blue), with excluded residues in white. The CDR regions are indicated in magenta denoted as CDR1, CDR2 and CDR3 and are placed at the top of each loop.

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Top view Figure S16: Average structures obtained during each of the 10 parallel trajectories at 400 K for 5sv4 colored by the average per-residue Q-value (0 to 1: red to blue), with excluded residues in white. The CDR regions are indicated in magenta denoted as CDR1, CDR2 and CDR3 and are placed at the top of each loop.

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Back view Top view Figure S17: Average structures obtained during each of the 10 parallel trajectories at 400 K for 3b9v colored by the average per-residue Q-value (0 to 1: red to blue), with excluded residues in white. The CDR regions are indicated in magenta denoted as CDR1, CDR2 and CDR3 and are placed at the top of each loop.

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Top view Figure S18: Average structures obtained during each of the 10 parallel trajectories at 400 K for 4tyu colored by the average per-residue Q-value (0 to 1: red to blue), with excluded residues in white. The CDR regions are indicated in magenta denoted as CDR1, CDR2 and CDR3 and are placed at the top of each loop.

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Back view Top view Figure S19: Average structures obtained during each of the 10 parallel trajectories at 400 K for the 4idl R71I mutant colored by the average per-residue Q-value (0 to 1: red to blue), with excluded residues in white. The CDR regions are indicated in magenta denoted as CDR1, CDR2 and CDR3 and are placed at the top of each loop. The residues involved in our mutations (Asn27, Ile71) are shown, colored in cyan and denoted in black text.

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Top view Figure S20: Average structures obtained during each of the 10 parallel trajectories at 400 K for the 4idl N27D mutant colored by the average per-residue Q-value (0 to 1: red to blue), with excluded residues in white. The CDR regions are indicated in magenta denoted as CDR1, CDR2 and CDR3 and are placed at the top of each loop. The residues involved in our mutations (Asp27, Arg71) are shown, colored in cyan and denoted in black text.

Front view
Back view Top view Figure S21: Average structures obtained during each of the 10 parallel trajectories at 400 K for the 4idl R71I/N27D mutant, respectively colored by the average per-residue Q-value (0 to 1: red to blue), with excluded residues in white. The CDR regions are indicated in magenta denoted as CDR1, CDR2 and CDR3 and are placed at the top of each loop. The residues involved in our mutations (Asp27, Ile71) are shown, colored in cyan and denoted in black text.