Horwich was just a boy, growing up in a western suburb of Chicago, when Anfinsen conducted his famous experiment. But he remembers the day he learned about it, the day Anfinsen received the Nobel Prize in 1972. Horwich was an undergraduate at Brown University, having joined the first class of students in a program that combined an undergraduate degree with a medical degree. That evening, he says, “we went to the lab and looked for every protein we could find to duplicate Anfinsen’s experiment, it was so astonishing.” Taken as he was by Anfinsen’s discovery, Horwich could not have predicted that decades later his own work would forever amend it.

Horwich completed medical school, graduating first in his class at Brown, followed by a residency in pediatrics at Yale School of Medicine. He loved the human contact that came with clinical medicine but knew the life of a physician wouldn’t satisfy his curiosity. Lured by the California climate and a chance to study a particular virus, polyoma, with Walter Eckhart and Tony Hunter, he took a postdoctoral research position at the Salk Institute before returning to Yale in 1981. There, in the lab of Leon Rosenberg, Horwich began his work on a human enzyme—ornithine transcarbamylase (OTC)—that would prove key to overturning the dogma, grounded in Anfinsen’s work, that proteins fold on their own.

“In whatever direction the project needs him to go to become expert, he does that. It’s one of the hallmarks of a great scientist, following the trail wherever it leads.”

Richard Lifton

OTC facilitates the conversion of ammonia to urea in human cells, neutralizing waste. In newborns with a rare, X-linked genetic mutation, OTC deficiency can cause ammonia to accumulate. Seemingly normal at birth, the infants can fall into a coma within days. Rosenberg, Horwich, and colleagues cloned and sequenced the gene responsible for OTC, ultimately developing a genetic test that allowed patients with a family history of the disease to determine whether a fetus carries the often lethal mutation.

Capturing a Complex Picture

Horwich established his own lab at Yale and was soon joined by Krystyna Furtak, his technical “right hand” to this day, and graduate student Ming Cheng. They had learned that to do its job, OTC must first be delivered to the mitochondria, the oval organelles that supply energy to cells. Curious about how this happens, the group inserted the human OTC gene into yeast. They then created mutant forms of the yeast, looking for failures in the OTC pathway that might help them decipher the steps involved.

By then it was known that proteins generally can’t enter mitochondria in their bulky three-dimensional forms. To pass through tiny entryways in the mitochondrial membranes, they must first unfold. Once inside, before they can do their jobs, they must fold up again. One evening in 1987, after a day of examining mutant yeast for variations, Horwich and Cheng, looked across the lab bench at each other and asked a question no one else had: might the OTC protein need help folding after it has entered the mitochondrial chamber?

Anfinsen’s experiment, after all, had been conducted in the protected isolation of a test tube; the environment inside a cell is a cacophony of enzymes, chemicals, and tiny protein machines. Some of these machines were known to help refold proteins under stressful conditions that disrupt their shape, such as heat. What if such machines were also required for a protein to fold under normal conditions? Horwich and Cheng decided to look for a mutant yeast strain in which OTC made it into the mitochondria but didn’t fold properly once there. That, they guessed, would signal the absence of any such protein-folding machine if it existed.

Within days, Cheng had identified such a mutant, which seemed to confirm their hypothesis. Hartl, an expert on how proteins are imported into the mitochondria, then at the University of Munich, heard about these experiments and called Horwich to see if he needed assistance on the biochemistry side of the problem. “And of course we did,” laughs Horwich. “We were three people in a new lab who had little or no experience with yeast biochemistry and were stumbling our way around."

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