What lies beyond uranus? Preconceptions, ignorance, serendipity and suppressors in the search for biology's secrets.

DISULFIDE bonds in proteins—how these bonds are formed, how they are cleaved, and how they participate in protein folding—have become my passion over the past 25 years. During this period, the discoveries in my lab in this very biochemical realm have come largely from genetic approaches. Many factors have influenced the pace of our progress, including at times ignorance of certain biochemical dogma and at other times unreflective adherence to such dogma. We have been impelled by the conviction that well-thought-out genetic approaches can yield insights into how processes of protein chemistry occur in a living cell. Yet, despite our carefully constructed ‘‘rational’’ approaches, serendipity, a workhorse of science, has consistently led us in unexpected directions. Disulfide bonds, the covalent bonds between sulfurs of cysteine residues, contribute to the folding, structure, and stability of many proteins. In gram-negative bacteria, structural disulfide bonds are found only among those proteins translocated through the cytoplasmic membrane such as secreted toxins, components of appendages such as flagella, many periplasmic proteins, and the periplasmic domains of some outer membrane and cytoplasmic membrane proteins. In eukaryotic cells, proteins with stable disulfide bonds are among the proteins that pass through the endoplasmic reticulum. They include secreted proteins and the extracytoplasmic domains of plasma membrane proteins. Few, if any, proteins with structural disulfide bonds are located in the cytoplasm, whether in eukaryotes or prokaryotes. However, certain cytoplasmic reductive enzymes that use the redox chemistry of cysteine in their active sites do form disulfide bonds as part of their catalytic cycles, but these bonds are subsequently reduced to regenerate active enzyme. For many years, the accepted explanation for the specialized subcellular location of proteins with disulfide bonds was based on a simple view: The periplasm of bacteria, because it is exposed to oxygen, and the lumen of the endoplasmic reticulum, perhaps because of the presence of oxidized glutathione, are oxidizing environments. Thus, the formation of disulfide bonds in proteins, an oxidative step, takes place in such environments without need for any enzyme catalysts. In contrast, the cytoplasms of both eukaryotic and prokaryotic cells are reducing environments maintained by electrons transferred from molecules such as NADH, NADPH, and reduced glutathione. Either disulfide bonds cannot form under these reducing conditions or, if they do, they are converted back to free cysteine residues by the reducing environment. It seemed as though any further exploration of these processes was unnecessary; the explanations were at hand. The assumption that disulfide bonds form spontaneously in an oxidizing compartment derived directly from the important experiments of Anfinsen et al. (1961) on protein folding in the early 1960s. They showed that when bovine pancreatic ribonuclease, which contains four disulfide bonds, was reduced and denatured, it could reassemble into its active structure in the test tube in the presence of oxygen and in the absence of any enzyme catalysts. These findings suggested that no such catalysts for the oxidative folding process should be necessary in vivo. Nevertheless, the kinetics of disulfide bond formation in ribonuclease in these experiments was very slow, incommensurate with the rapid kinetics that we now know occurs in vivo. Furthermore, a significant fraction of ribonuclease folded into a non-native conformation with the ‘‘wrong’’ cysteines joined in disulfide bonds. This latter finding led Anfinsen and coworkers to predict the existence of and then to find the enzyme, protein disulfide isomerase (PDI), that promoted rearrangement of the disulfide bonds of incorrectly folded ribonuclease into the native conformation (Goldberger et al. 1963). They did not see the necessity of looking for an enzyme that catalyzes disulfide bond formation itself. We became interested in the issue of disulfide bond formation during the course of our studies on translocation of proteins across the cytoplasmic membrane Address for correspondence: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. E-mail: [email protected]