Objective To investigate the impact of the dominant deafness point mutation p.D179N on the oli-gomeric equilibrium state of Connexin 26(Cx26).Methods The wild-type Cx26 fusion protein(Cx26-WT-GFP)and mutant fusion proteins(Cx26-D179N-GFP,Cx26-D179C-GFP)were expressed in HEK293F cells.By using Fluorescence-detection size-exclusion chromatography(FSEC)and size-exclusion chromatography(SEC)to analysis the oligomeric state of the target protein based on malecular weight under the condition of solubilization and purifica-tion respectively.Cryo-electron microscopy(Cryo-EM)single particle analysis(SPA)was conducted to analysis the target protein's oligomeric states based on the 2D classification morphology of the protein particles.Results In vitro,the wild-type Cx26 protein(Cx26-WT)is almost exclusively dodecameric.The deafness mutation p.D179N protein(Cx26-D179N)exists as both dodecamers and hexamers,whereas the artificial mutation p.D179C protein(Cx26-D179C)does not form dodecamers.Conclusion The dominant deafness mutation GJB2 p.D179N could weaken the ability of docking between hexameric proteins,which could affect the balance between hexamers and do-decamers.

Objective To investigate the impact of the dominant deafness point mutation p.D179N on the oli-gomeric equilibrium state of Connexin 26(Cx26).Methods The wild-type Cx26 fusion protein(Cx26-WT-GFP)and mutant fusion proteins(Cx26-D179N-GFP,Cx26-D179C-GFP)were expressed in HEK293F cells.By using Fluorescence-detection size-exclusion chromatography(FSEC)and size-exclusion chromatography(SEC)to analysis the oligomeric state of the target protein based on malecular weight under the condition of solubilization and purifica-tion respectively.Cryo-electron microscopy(Cryo-EM)single particle analysis(SPA)was conducted to analysis the target protein's oligomeric states based on the 2D classification morphology of the protein particles.Results In vitro,the wild-type Cx26 protein(Cx26-WT)is almost exclusively dodecameric.The deafness mutation p.D179N protein(Cx26-D179N)exists as both dodecamers and hexamers,whereas the artificial mutation p.D179C protein(Cx26-D179C)does not form dodecamers.Conclusion The dominant deafness mutation GJB2 p.D179N could weaken the ability of docking between hexameric proteins,which could affect the balance between hexamers and do-decamers.