Disulfide bonds constrain the polypeptide backbone and reduce conformational variability in proteins. The blood clotting protein fibrinogen is constitutively produced as multiple partially disulfide-bonded states, suggesting that individual fibrinogen molecules have a variety of conformational forms. This hypothesis was tested by resolving fibrinogen molecules on beads coated with different fibrinogen ligands and measuring their disulfide states by differential cysteine alkylation and mass spectrometry. Polyclonal anti-fibrinogen antibodies resolved states where all 11 measured disulfides across the molecule were significantly less formed. In contrast, the GHRP peptide, which binds the fibrinogen β-nodule, resolved states in which seven β-nodule and central E region disulfides were significantly more formed, while the GPRP peptide, which binds the γ-nodule, resolved states in which only one disulfide was significantly more formed. To probe the link between disulfide state and conformation, in silico analysis of all 32 possible disulfide-bonded states of the β-nodule revealed that the conformational flexibility of this domain and predicted GHRP interactions in its binding pocket are predicated on the oxidation state of one of the five β-nodule disulfides, βC424-βC437. These findings indicate that the different disulfide-bonded states of fibrinogen adopt an extensive array of conformations that are selectively recognized by different fibrinogen ligands.
Pijning et al. (Mon,) studied this question.