Abstract
Disulfide bonding of cysteines is one of the most important protein modifications, and it plays a key role in establishing/maintaining protein structures in biologically active forms. Therefore, the determination of disulfide bond arrangement is one important aspect to understanding the chemical structure of a protein and defining its functional domains. Herein, aiming to understand how the HIV-1 envelope protein's structure influences its immunogenicity, we used an MS-based approach, liquid chromatography electrospray ionization Fourier transform ion cyclotron resonance (LC/ESI-FTICR) mass spectrometry, to determine the disulfide linkages on an oligomeric form of the group M consensus HIV-1 envelope protein (Env), CON-S gp140 ΔCFI. This protein has marked improvement in its immunogenicity compared to monomeric gp120 and wild-type forms of gp140 Envs. Our results demonstrate that the disulfide connectivity in the N-terminal region of CON-S gp140 ΔCFI is different from the disulfide bonding previously reported in the monomeric form of gp120 HIV-1 Env. Additionally, heterogeneity of the disulfide bonding was detected in this region. These data suggest that the V1/V2 region does not have a single, conserved disulfide bonding pattern and that variability could impact immunogenicity of expressed Envs.