Figure 2.
Protein modeling, dynamics simulation, and functional studies. (A) Evolutionary conservation analysis mapped onto the predicted protein structures. (B) The effect of HMCN1 variants on protein stability was calculated by FoldX (ΔΔG). All three variants are predicted to destabilize the protein substantially. (C) A scheme of an Ig tandem used for MD simulations is presented (left panel). Each of the Ig domains bearing a variant along with the two flanking Ig domains was modeled (with and without the deleterious variant) and used to run 250-ns MD simulations while measuring the interdomain angle as indicated in the left panel. The frequency distribution of the interdomain angle along the simulation trajectories is shown. Black curves denote the WT proteins, while the red curves denote the mutants as indicated. Note that the amino acid substitution causes significant deviations from the straight axis of the constructs harboring the Ig38 and Ig40 Ig domains compared to WT. (D) Structural studies of hemicentin-1 Ig27 domain are presented (each experiment was repeated three times). The left panel shows the elution profiles from size-exclusion chromatography for Ig27 WT (black) and mutant (red) domains. Inset, SDS-PAGE of purified Ig27-WT and Ig27-Gly2939Ser; molecular masses in kDa are indicated to the left. Note that the mutant domain shows an apparent larger molecular weight compared to WT. The right panel shows tryptophan fluorescence analysis of Ig27 WT (black) and mutant (red) domains, revealing a significant shift in emission wavelength and amplitude of the mutant domain as compared with the WT domain. (E) Purified Ig38 and Ig40 WT and mutants (p.Ala3969Thr and p.His4084Tyr, respectively) domains were analyzed by size exclusion chromatography (each experiment was repeated three times). Inset, SDS-PAGE analysis; molecular masses in kDa are indicated to the left. Note the reduced amount of Ig38 and absent Ig40 protein, as corroborated by the nickel exclusion chromatography data (Fig. S1). Source data are available for this figure: SourceData F2.

Protein modeling, dynamics simulation, and functional studies. (A) Evolutionary conservation analysis mapped onto the predicted protein structures. (B) The effect of HMCN1 variants on protein stability was calculated by FoldX (ΔΔG). All three variants are predicted to destabilize the protein substantially. (C) A scheme of an Ig tandem used for MD simulations is presented (left panel). Each of the Ig domains bearing a variant along with the two flanking Ig domains was modeled (with and without the deleterious variant) and used to run 250-ns MD simulations while measuring the interdomain angle as indicated in the left panel. The frequency distribution of the interdomain angle along the simulation trajectories is shown. Black curves denote the WT proteins, while the red curves denote the mutants as indicated. Note that the amino acid substitution causes significant deviations from the straight axis of the constructs harboring the Ig38 and Ig40 Ig domains compared to WT. (D) Structural studies of hemicentin-1 Ig27 domain are presented (each experiment was repeated three times). The left panel shows the elution profiles from size-exclusion chromatography for Ig27 WT (black) and mutant (red) domains. Inset, SDS-PAGE of purified Ig27-WT and Ig27-Gly2939Ser; molecular masses in kDa are indicated to the left. Note that the mutant domain shows an apparent larger molecular weight compared to WT. The right panel shows tryptophan fluorescence analysis of Ig27 WT (black) and mutant (red) domains, revealing a significant shift in emission wavelength and amplitude of the mutant domain as compared with the WT domain. (E) Purified Ig38 and Ig40 WT and mutants (p.Ala3969Thr and p.His4084Tyr, respectively) domains were analyzed by size exclusion chromatography (each experiment was repeated three times). Inset, SDS-PAGE analysis; molecular masses in kDa are indicated to the left. Note the reduced amount of Ig38 and absent Ig40 protein, as corroborated by the nickel exclusion chromatography data (Fig. S1). Source data are available for this figure: SourceData F2.

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