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MICROBIOLOGY
Early Step in Protein-folding Revealed by Bacterial MutantThe bending of ribbons of amino acids into complex and curlicued proteins is one of nature's most striking sleights of hand. It is also one of the most consequential. The proper functioning of the body depends on proteins assuming exquisitely exact shapes. Indeed, mistakes in protein folding are thought to play a role in a wide variety of illnesses from Creutzfeldt-Jakob disease, the human equivalent of mad cow, to Alzheimer's and Parkinson's diseases. Jon Beckwith, Hiroshi Kadokura, and their colleagues have characterized an early step in protein folding, a finding that could someday prove useful in therapeutic attempts to combat such disorders.
DsbA promotes protein folding in bacteria by donating a disulfide bond (S-S). The process begins when a string of amino acids moves into the pericellular space (left). The protein binds DsbA, exchanging a hydrogen atom for a disulfide bond, which it then uses to fold and stabilize itself (right). Normally, DsbA and the protein dissociate too quickly for the intermediate form, or mixed disulfide bond, to be observed. Certain mutants slow down the process, allowing the mixed disulfide complex to be observed (middle). (Image by Jeff Cleary) For years, researchers thought that the entire set of instructions for these feats of molecular origami lay in a protein's amino acid sequence. In 1991, Beckwith and his colleagues showed that even in simple bacteria, the folding of certain proteins, in particular those located outside the cell, requires an external agent. The agent, DsbA, was thought to work its transformational magic by bestowing upon proteins an all-purpose molecular hook, or disulfide bond (the origin of the abbreviation Dsb). The protein could then use it to weave together and stabilize its three-dimensional structure. Yet in all this time, no one has been able to glimpse DsbA in the act of binding, let alone bequeathing a disulfide bond to a protein substrate.
"This is the most exciting thing that has happened in my lab in years," said Beckwith, the American Cancer Society professor of microbiology and molecular genetics at HMS. "One of the things I am happiest about is that in this day of DNA manipulation, this in vivo genetic approach is still important for solving biological problems." Pokey ProteinsThe bacterial mutants causing all the commotion belong to a special class. "Some mutants slow down special steps or enzyme reactions so we can see what we cannot usually see," said Kadokura, instructor in microbiology and molecular genetics at HMS. Normally, the binding of extracellular proteins to DsbA is a fleeting affair. Strings of amino acids produced inside the cell move through the membrane into the pericellular space where they fasten onto one of DsbA's disulfide bonds. DsbA instantly dissociates from the protein, leaving its bond behind; it receives a new one from another protein, DsbB.
A discovery by Jon Beckwith (left), Hiroshi Kadokura, and colleagues could lead to a better understanding of how proteins are misfolded in diseases such as Parkinson's, Alzheimer's, and Creutzfeldt-Jakob. (Photo by Phil Farnsworth) With Hongping Tian, now an HSPH graduate student, Kadokura and Beckwith used ultraviolet radiation to create more than a million bacterial mutants, one of which had a remarkable form of DsbA. Usually when a protein is run on a western blot gel, which separates proteins by molecular weight and electrical charge, it forms a uniform band. But the mutant DsbA produced many spots, each representing a slightly different molecular weight and all of them much higher than the wild type version. The reason the mutant DsbA was so heavy is that it was still bound to its substrate--not just one, but a variety of proteins, several of which were known DsbA partners. Importantly, they appeared to be joined by DsbA's disulfide bond. "When we broke the disulfide bond with a reducing agent, the mutant DsbA collapsed back into the DsbA band at the bottom," said Kadokura. He, Beckwith, and colleagues, who were funded by the NIH, believe that the mutant's single amino acid change, from proline to threonine, interferes with DsbA's ability to quickly dissociate from its protein substrate. Intriguingly, the researchers found another mutant carrying a change in the same proline, but in this case, it became a serine. It, too, appeared to get stuck, but to DsbB rather than the protein substrate. "Subtle changes in the same proline could affect its interaction with either substrate or DsbB," said Beckwith. Bond YieldAs it turns out, disulfide bonds play a role in many physiological processes, not just protein folding. "Disulfide bonds are present in a lot of important proteins, like insulin, antibodies, hormones, and membrane receptors," Beckwith said. Attempts to create therapeutics from these proteins could benefit a variety of diseases, including those in which protein misfolding is the culprit. "If you want to engineer proteins or sequences of proteins that have disulfide bonds and have them form efficiently, you might want to engineer them to have proper amino acid sequences around these sites," he said. "But that is way down the line."To get to that point will require a much more thorough understanding of how exactly DsbA transfers its disulfide bonds. Beckwith relishes the challenge. "I like getting to the depths of a problem," he said. "What makes it exciting is that we are able to use bacterial genetics to get at disulfide bonds. We are doing microarrays without having to do microarrays." --Misia Landau |
