Functional Disulphide Bonds

Abstract

Disulfide bonds are the covalent links between the sulfur atoms of pairs of cysteine amino acids in the polypeptide chain. They are the second most common covalent bond linking the polypeptide backbone after the peptide bond. For several decades, disulphide bonds were thought to be inert in the mature protein. We know now that a subset of disulfide bonds control the function of the mature protein in which they reside when cleaved and/or formed by different factors. These allosteric disulphides and the factors that cleave them have been shown to control protein function across biological systems and life-forms. This book describes the current techniques used to study this post-translational modification and to exploit it for therapeutic outcomes. To set the scene, how disulphide bonds are classified and how to study their evolution are presented. The following chapters describe the protein chemical, molecular dynamics, and force spectroscopy techniques used to identify functional disulfides and/or study their behavior. The book then focuses on techniques employed in the study of the factors that cleave and/or form disulphide bonds. The redox state of disulphide bonds is influenced by small molecules that alkylate the unpaired cysteine thiols of the reduced disulphide. The techniques used to study these cysteine modifications are highlighted. Due to their inherent reactivity, the cysteine thiol is an attractive target for covalent drugs. An approach to identifying specific inhibitors of protein cysteines is described. Three examples of how particular functional disulphides are assayed in mouse and human tissues are then described in some detail. These methods will be translatable to other functional disulphides. Finally, small molecule probes that react with the reduced forms of functional disulfides are described. These probes are being used to assess prothrombotic potential in humans and for noninvasive imaging of tumor cell death in cancer patients.