Making the Gradient

The young Kaback obtained the mutant strains from Maas and, in his spare time, started working on preparing membranes from the bacteria. “I worked all my weekends and holidays on these membranes—all the way through med school,” he says. “I’d get a little bit of transport, and there was always a difference between wild-type E. coli and the mutants. So I was determined to stick with it.”

Not everyone was so enthusiastic about his idea. “I used to go to honors lectures on Saturday mornings at NYU where they would bring in these real big shots,” says Kaback. “And one week, Werner Maas arranged for me to have an audience with Francis Crick before the start of his lecture. I was ushered into the room, scared to death. But I told him about my work. Sir Francis listened patiently and, looking down his nose, said, ‘No, no. That can’t possibly be right.’”

“I remember thinking to myself, ‘What the hell does this guy know about transport?’ So I kept working on my membranes,” says Kaback, who ultimately discovered that Crick was, in fact, correct. “But that’s what science is all about. You come up with some idea, do some experiments, and eventually your idea turns out to be total baloney. But along the way, you’re taken in a different direction that turns out to be much more important than the idea that originally got you into the whole thing.” In Kaback’s case, that early misconception about RNAs and amino acid transport led him to a raft of discoveries about how cells use proteins—such as the lactose permease of E. coli (encoded by the well-studied lacY gene)—to move sugars and other key molecules across their membranes. Here he reviews some of those findings, and talks about why intelligence is overrated and where he’d like his brain to head next.

Fort Bethesda. In 1964, Kaback headed to the National Institutes of Health—instead of Vietnam. “If you were really lucky, and had all the right credentials and knew the right people, you could spend your selective-service time doing research at NIH—with no uniforms, no saluting, and no bullets sailing over your head or bombs blowing up under your feet,” he says. “Actually, the Vietnam War made NIH the greatest biomedical research institution in the world.” Investigators there had their pick of the litter when it came to filling their labs with American talent. With a recommendation from the elder Al Gilman, then chair of pharmacology at Einstein, Kaback wound up in the laboratory of enzymologist Earl Stadtman. “Stadtman had a passing interest in transport mechanisms, so he kindly let me bring my own problem to his lab.” After two years at NIH, Kaback was offered a position at the University of California, Irvine. But he turned the job down when Stadtman asked him to stay. “I didn’t even ask if I would get a technician,” says Kaback. “I just loved being at NIH because all I had to do was science. I could play with my membrane vesicles day and night. And I really became a scientist there.”

To JBC and back. In 1962, Kaback submitted his first paper to the Journal of Biological Chemistry. “It was the first year that JBC accepted ‘preliminary communications,’” he says. “And I got back a letter saying that the work was of such potential importance that I should do more experiments and publish it as a full paper. With this note came 20 pages of suggested experiments from reviewers. I didn’t know how the system worked. So I did the experiments. All of them. And I submitted the paper again. They sent me back more pages of suggested experiments. This went on for about seven years,” he says. By then, Kaback had amassed enough data that his “preliminary communication” had become two full papers—neither one any closer to publication. “Finally, one reviewer remarked that the first paper wasn’t so interesting anymore, and suggested I submit it as a preliminary communication. Well, I went a little nuts. I called the editor of JBC—Rockefeller’s William Stein—and started screaming. He let me keep going until I ran out of steam and then he gently advised me to go through the comments, one by one, and either agree—and do another experiment—or explain why I disagreed. It took me a while, but I sent back a 20-page rebuttal, single-spaced. Two weeks later, both papers were accepted without revision.”

A ticket to Mitchell land. When Peter Mitchell proposed his theory of chemiosmotic coupling in the 1960s, Kaback was one of the last to believe him. “Mitchell had this insane hypothesis that ATP production was driven by an electrochemical proton gradient,” says Kaback. “Nobody understood what he was talking about. So my mind-set was against this to start with. Also, I was trained as a pediatrician. So what did I know about, well, anything, really?” Imagine Kaback’s surprise when his membrane vesicles proved Mitchell was right. Kaback was using his vesicle prep to try to figure out how the sugar lactose gets into cells. If Mitchell was correct, the transport of the sugar into vesicles (and cells) must be powered by an electrochemical proton gradient. But no one had been able to measure a difference in proton concentration across bacterial membrane vesicles. Then Kaback started playing with a technique called flow dialysis, which had been introduced to his lab (at this point at the Roche Institute of Molecular Biology) by postdoc Gary Rudnick. The approach—which allowed Kaback and his colleagues Sofia Ramos and Shimon Schuldiner to precisely measure proton concentration gradients without disrupting the vesicles—“showed there was a gigantic electrochemical proton gradient,” he says, “and that this gradient was driving transport.” Moving lactose or other substrates across the membrane consumed some of this electrochemical energy, a phenomenon Kaback et al. could also quantify. “It was hard to argue against Mitchell with that kind of data. So in the end it’s really simple. The whole issue is: how much energy do you have stored in this electrochemical ion gradient, and is that enough energy to do the work of accumulating substrate against, say, a hundredfold concentration gradient? And we could show all these things.”

The hard part. Making the vesicles, says Kaback, “is easy. Most membranes form vesicles when they tear. Because if you have hydrophobic ends in an aqueous solution, which is thermodynamically unstable, they want to reseal. The trick is getting them to transport.” Kaback discovered that D-lactate—one form of lactic acid, a small molecule unrelated to the sugar lactose—can supply the energy to get the job done. Attached to the cytoplasmic side of E. coli membrane vesicles is an enzyme, D-lactate dehydrogenase, which oxidizes D-lactate and hands its electrons to the molecular machinery that generates a proton gradient. In early experiments, that gradient fueled the uptake of a radioactive metabolite at such a furious pace that it broke the anode wire Kaback was using to measure the radioactivity.

Trading Cys-es. To probe the three-dimensional structure of lactose permease (LacY)—the protein responsible for bringing lactose into E. coli—Kaback devised a form of site-directed mutagenesis called cysteine-scanning mutagenesis. “We literally replaced each of the 400-plus residues in the protein, one at a time, with a cysteine,” he says. The approach yielded several different kinds of information. First, it revealed a handful of residues that were critical for transport activity: replacing them with cysteine (or other residues) killed transport. Next, adding cysteines at various sites around the protein gave Kaback and company places to attach fluorescent probes and other chemical labels that marked different segments of the molecule for structural analysis. Finally, because cysteines can be coaxed into forming crosslinks with one another if they’re close enough, mutants containing cysteine pairs could be used to assess whether one helix lies next to another. Examining these mutants in the presence and absence of a lactose homologue—and ultimately combining this data with the 3-D crystal structure of the protein—Kaback and colleagues determined that LacY moves lactose across the membrane when its two halves rock back and forth against one another, alternately exposing its sugar-binding pocket to the one side of the membrane (where it picks up a proton and lactose) and the other (where it releases them). “The whole project took about ten years,” says Kaback, who by then was an HHMI investigator at UCLA. “Over that period, there must have been 50 people involved. Students would come to lab wanting to learn how to do site-directed mutagenesis, so we’d give them a helix and say, ‘Go to it.’” And having the Hughes funding made the whole thing possible. “Cysteine scanning is now a very popular technique,” says Kaback. “Everyone uses it. But it’s a good example of something an NIH study section would never have OK’d. Never.”

Seeing is believing. “Everybody thinks DNA is the biggest finding in modern biology. And it is. But equally as big is bioenergetics and chemiosmosis—Peter Mitchell’s ingenious concept. Chemiosmosis is very important, but it’s difficult to grasp because you can’t crystallize or isolate electrochemical ion gradients. You can only measure them. DNA you can see. And that’s a big difference, because people believe what they can see. But electrochemical ion gradients: what the hell are they?”

Dr. Picasso. “There’s a creative aspect to science, the same as painting or composing music. Philosophers can argue that scientists don’t really create things, they discover what is already there. But it’s not the discovery itself that’s creative, it’s the style with which the experiments are done.”

The evils of R01s. “The act of writing grants is not only a waste of time, I think it’s anti-intellectual. When you write a grant, you’re not coming up with new ideas; you’re trying to sell something to a study section. To me, that’s not what science is about.”

The 3 percent solution. “One of the reasons I voted for Obama is because he said he would put 3 percent of the gross national product into science. If the economy improves, I think he should really try to put his money where his mouth is, because we are not the first in the world in biological sciences anymore. We have to get back to a situation where we’re not funding just 15 percent of the grant proposals submitted, or we’re going to end up losing scientists at an increasing rate.”

The Dark Side. “I no longer flatly advise my postdocs to go into academia. I tell them, ‘Look, if you want to be an assistant professor, you have to be really hungry for it.’ The average age for getting a first grant is 42 years old. That’s absolutely terrible. So I tell people, ‘Unless you are really, really willing to sacrifice most everything else to your scientific career, go into biotech. But remember, when you go to the Dark Side, the thing you give up is your freedom.’”

Character profile. “To be a good scientist, I think it helps to be smart—but not too smart. I think if you’re too smart you may talk yourself out of too many experiments. And you can’t be patient. Impatience is very important. You have to really want results. If you’re too patient, nothing will ever get done.”

Nothing for me. “I only eat once a day, like a dog. Three times a week, that one meal is a box of Slim Fast bars, which is like 600 calories. That’s on top of coffee and pills in the morning, and maybe a Diet Pepsi or two during the day. I’m 75 years old. So I guess the only reason I do this is to have a good-looking corpse.”

Last organ left standing. Kaback enjoyed skiing, tennis, and dancing before an accumulation of injuries sent him to the sidelines. “Football ruined my left knee. I tore my ACL in 1950-whateveritwas. That needs to be replaced. I’ve had both shoulders replaced. I’ve had surgery on my back, so these days I walk with a cane. I overdid it. I don’t do anything halfway. I would like to have my brain put in another body. An 18-year-old body. I don’t care which sex. But tall and thin. One of these bodies that doesn’t gain weight when it eats.”

It’s an honor. Kaback learned of his election to the National Academy while on a Caribbean cruise with his brother-in-law, a tire salesman. “Once a year, Bucky would take his top salesmen and his big customers on these expensive cruises. In 1987, he invited my wife and me.” During the trip, Kaback got the good news. “My wife and I were all excited. But we were on a ship with 500 tire salesmen. None of them knew what the National Academy was. When my brother-in-law announced I’d been elected, my father-in-law asked, ‘Do you get free tickets to the Philadelphia Academy of Music?’”

Netflix has it. “My all-time favorite movie is All That Jazz. It’s the only movie I know of where there’s a really beautiful dance number done to open heart surgery.”