DISULFIDE STRUCTURE OF BIOMEDICALLY IMPORTANT PROTEINS

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Disulfide bonds are a critically important determinant of the shape and, thus, the activity of some biomedically important proteins. The common bleeding disorder, von Willebrand Disease, appears to be related to a defect in the disulfide bonding pattern among 18 cysteines (including two pairs of adjacent cysteines) of a particular protein (VWF) that disrupts the normal blood clotting cascade. Very little is known about the cysteine status of the von Willebrand protein (VWF), and such knowledge will help explain the cause of this disorder at the molecular level; however, VWF is resistant to the conventional proteolytic approach to disulfide mapping. Knowledge of the disulfide bonding pattern in the receptor- binding proteins for TGF-beta will help provide an important 'template' for the development of drugs (antagonists) for treatment of fibrotic disorders ,e.g., Duchennes muscular dystrophy; similarly, the development of other drugs (agonists) may serve as anti-cancer agents by promoting the negative proliferative response to TGF-beta. However, the highly knotted, cysteine-rich (up to 12 cysteines, 3 of which are adjacent) receptor-binding proteins for TGF-beta are resistant to conventional disulfide mapping. Developing a protocol for the disulfide mapping of VEGF homodimer will provide the basis for designing and monitoring the proper folding of related pharmaceutical proteins with angiogenic activity. Our novel approach to disulfide mapping, based on cyanylation of and cleavage at cysteine residues, offers new hope for determining the disulfide bonding pattern of the biomedically important cystinyl proteins described above that are refractory to conventional methodology. Cyanylation is selective for free sulfhydryls and can be accomplished at pH 3, a condition that suppresses problems with disulfide scrambling. We have demonstrated that the cyanylation/cleavage approach is applicable to proteins containing adjacent cysteines, an attribute that recommends it for successfully attacking the difficult analytical challenges posed by the proteins described herein. An a1gorithm will be developed to assign the connectivity of cysteines in disulfide bonds given an input of amino acid sequence and mass spectra of cyanylation/cleavage products.