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Johns Hopkins UniversityArts and Sciences Magazine
Biophysics Al dente

Illustrations by Luc Normandin


Imagine you have a really long noodle of spaghetti in a pot of boiling water. You put the lid on. You cook it for a while. You come back. You open the lid and peer through the steam.

The noodle has spelled your name.

In other pots, even longer noodles are performing more fantastic feats of organization: One creates your grocery shopping list; another, the Preamble to the Constitution. How, you wonder, did something so incredible take place? "That's the protein-folding problem," professor Doug Barrick exclaims as he ponders quintessential biophysics questions in class and in his Jenkins Hall lab on the Homewood campus.

"How," Barrick marvels, "do the noodles know? They do know."

Doug Barrick and his lab are pushing to create repeat proteins that are remarkably stable and thus, he says, “better than nature.”

Photo: Mike Ciesielski

Barrick, who earned extra cash in high school as a pasta maker, explains to his students that the mysteriously folding noodle is analogous to a natural phenomenon even more exquisite than it is ubiquitous: Right now, deep inside our cells, linear sequences of chemicals known as amino acids are assembling themselves precisely into three-dimensional molecules known as proteins, the folded shapes of which make cellular sense in that they determine function. When a protein mis-folds, chaos ensues. Cells die or multiply when they aren't supposed to, resulting in all manner of diseases and disorders. That's an aberration, though. Most times, they do fold just right.

Mario Amzel, director of Biophysics and Biophysical Chemistry at the Johns Hopkins School of Medicine, points out that protein folding has been one of the issues at the frontier of biophysics for as long as he has been working in the field.

How long is that?

"Probably more than you are alive," he laughs, twisting a length of his salt-and-pepper eyebrow. "Very long. Very long."

He's being awfully cagey for someone whose daily mission is to be quantitative. I try again: "How long have you been at Hopkins?"

More evasive laughter. Still no numbers, however. Amzel refuses to be pinned down, offering only the "join-the-club" smile of one who's learned to live with the fact that not all questions get answered just for the asking.

"A question of mine involves predicting function from the structures of proteins," he says. "If function is completely reliant on structure, then behavior should be predictable. The nice thing would be to be able to predict behavior. But we are not there, not even close.

"We [biophysicists] don't work on finding a single fact that is related to a single case. We are discovering how nature works. It takes years to even imagine an answer. The answers we do get are just an infinitesimal part of the answer we are looking for."

It's Amzel's habit to indulge in regular constitutionals: Twice daily he escapes to the city streets, strolling and smoking his pipe to ponder Big Questions in peace, where few could understand what he was talking about if he happened to think out loud. He long ago learned to accept the fact that even his mother didn't have the first clue about his life's work.

"I would bring her to the lab, and she was amazed and amused, but that was it," Amzel recalls. "But she trusted me."

When Amzel says it would be great to be able to understand the folding mechanism and predict the structure of a protein from its amino acid sequence, he speaks for not only a select few scientists who straddle disciplines, but also the rest of us.

Why is it so important? Because knowing could ultimately hold clues for solutions to the world's energy problems. And cures for cancers, among other things.

"There are huge practical implications," Barrick says. "But in addition, it's a fascinating problem of self-organization. Given the complexity of protein structure, an astronomical number of conformations could be adopted. But they're not all adopted, only a very, very particular subset. Somehow, order comes out of chaos. And where and why, what the rules are, how that self-organization appears…. That's a beautiful problem."


More comprehensible than protein folding—if infinitesimally so—is how a handful of traditionally disparate fields at Hopkins folded together to give shape to the discipline now known as biophysics.

The term was coined at this university in 1949 by then-president Detlev Bronk, whose decree established the first-ever formal college biophysics course and department. At the time, the senior member of the new biophysics faculty was Haldan Keffer Hartline, who two decades later would win the Nobel Prize for discoveries relating to vision. Among the Arts and Sciences graduate students in the nascent field of biophysics was Paul Greengard. In Greengard's own Nobel autobiography (he was awarded in 2000 for work in signal transduction in the nervous system), he recalls, "I did my first laboratory research (in the new department of biophysics at Hopkins) under the supervision of Hartline."

One biophysics course morphed into four by 1950; six by 1951; and nine by 1954. The first bachelor's degree in biophysics was offered a couple of years later. What had allowed all this to take shape was a large bequest coinciding with Bronk's arrival. The gift came from the estate of May McShane Jenkins; it was ambiguously worded to encourage exploration of "water, heat, and light in the treatment of disease." Initially, the department was divvied up among the schools of Arts and Sciences, Medicine, and Hygiene/Public Health. Eventually, Public Health got folded in, leaving just the two, at Arts and Sciences and Medicine.

At this point, astute readers may well be wondering: What is biophysics, anyway? (This question tempts me to defer to the late, great Louis Armstrong who, when asked to define jazz, said: "Man, if you have to ask what it is, you'll never know.")

Biophysics is not jazz, however. Likewise, biophysics is not biology. And not physics. Nor is biophysics the sum total of the two disciplines.

Two driving forces in biophysics at Hopkins: Bertrand Garcia-Moreno and Juliette Lecomte.

Photo: Mike Ciesielski

Biophysics is described as "quantitative biology" because it pivots on the precision and predictive powers of math and computation as it seeks to answer biological questions. It applies the hard tools of physics to what was historically a softer, more descriptive science, explains Bertrand Garcia-Moreno, chair of Biophysics in the School of Arts and Sciences. Fundamental principles in statistical thermodynamics and kinetics help to reveal the highly choreographed molecular ballet going on inside of living cells: How do those interactions conspire to determine specific biological functions, and how do proteins and other biological molecules achieve their functional shapes? Who comes together when and for how long and with what exact force? Why and how do they separate, and at what speed to what distance?

"Cohesive" and "diffuse" aptly describe biophysics at Johns Hopkins. Each of the two independent but intensely collegial departments has a distinct flavor as well as separate budgets and chairmen: Garcia-Moreno and Amzel in the School of Arts and Sciences and the School of Medicine, respectively. And then there's an umbrella that shelters all: the 20-year-old graduate studies Program in Molecular Biophysics (PMB) directed by Arts and Sciences' Juliette Lecomte.

Funded by the National Institute of General Medicine at NIH, and recently renewed with a training grant of $6.5 million for five years, the program is widely regarded as the top biophysics program in the nation and perhaps in the world. The training grant annually supports about eight incoming graduate students who, after a year of rotating through a range of research labs, choose where they'll likely remain for the next half-dozen years.

“The students come from all different math and science backgrounds, and our job is to get them talking in each other’s languages. We have to teach the mathematicians to speak biology, the physicists to speak chemistry, and the biologists to speak computer science.”

—Jon Lorsch, admissions director for the graduate Program in Molecular Biophysics

Photo: Mike Ciesielski

"The students come from all different math and science backgrounds, and our job is to get them talking in each other's languages," says PMB admissions director Jon Lorsch, a self-described biophysical chemist based in the School of Medicine. "We have to teach the mathematicians to speak biology, the physicists to speak chemistry, and the biologists to speak computer science."
Portuguese scientist Eva Cunha is a multilingual fourth-year graduate student working in Barrick's lab. She meets with him regularly to discuss ongoing experiments related to a collaborative bio-fuels project that involves a mingling of various disciplines in the schools of Medicine and Arts and Sciences. Barrick's far-flung but close-knit accomplices include Jan Hoh in physiology, Justine Roth in chemistry, Eric Johnson in biology, Brendan Cormack in molecular biology, and Mario Amzel in biophysics.

During my visit, Cunha is attempting to embed cellulose-degrading enzymes in a synthetic template protein. She and Barrick pore over a series of bar graphs that represent the various activity levels of different enzyme preparations. They are talking CBMs, aka carbohydrate binding modules. Cunha is putting CBMs on her protein constructs so they'll anchor to the surface and produce more activity. High activity directly translates to ease with which biomass can be degraded into usable fuel precursors. "Put another way, it drops the cost of a gallon of product [fuel] because we have to use less of our valuable enzymes if they have more activity," Barrick explains.
The question at hand is one of synergy: Do combinations of enzymes strung together work better than when they are solo? Indeed, the latest data seem to indicate that at least one such grouping does show increased activity, a finding that Barrick declares "very exciting."

Even now, when "inter" is the buzz-prefix in academe and medicine, the PMB stands out as "the premier interdivisional and interdepartmental program university-wide, bar none," says Garcia-Moreno. Administered jointly by two departments across various divisions of the university, it's a bizarre model. Of 44 faculty who constitute the PMB, just seven are situated in Arts and Sciences' Biophysics Department. The majority are scattered about the university, medical school, and schools of Public Health and Engineering.

Like a folded protein, this arrangement works. Somehow.

"The program wouldn't be as good if we took a silo approach," Barrick says.
"We've got long tentacles."

“Ribo-switches are among our favorites. They’re little domains of the RNA that switch between two different shapes, depending on the presence or absence of a small molecule or ion.”

—David Draper
Vernon K. Krieble Professor of Chemistry

“Groupings of people that make sense intellectually may well have nothing to do with [academic] units," says David Draper, the Vernon K. Krieble Professor of Chemistry at the School of Arts and Sciences. "The fact that this program doesn't adhere to the traditional administrative boundaries reflects the state of the science." 
Draper, who directed the Program in Molecular Biophysics for 10 years, is based in the Chemistry Department at Homewood behind a door on which is posted a sign:  "Now Entering the RNA World. Authorized Persons Only."  
Draper studies ribonucleic acid (RNA), a "difficult" molecule that long was relegated to the backwaters of biophysics research, a sidelight in a field where the focus tends to be on proteins. (Vital for protein production, RNA is similar to DNA, kind of. One notable difference is that RNA in cells is usually single-stranded, while DNA is usually double-stranded.) An avid backpacker who is heading to Yosemite for an annual trek with several PMB colleagues, Draper says he was drawn to RNA research at least in part because he "always liked big white areas on the map."
Draper keeps an RNA zoo in his lab freezer. Each specimen in this collection of RNA domain sequences folds up into a specific shape; each has a specific biological function.
"Ribo-switches are among our favorites," Draper says, referring to a molecule that no one knew existed until eight years ago. "They're little domains of the RNA that switch between two different shapes, depending on the presence or absence of a small molecule or ion. These switches, which turn on or off the synthesis of specific proteins, respond to all kinds of standard molecules the cell needs to survive and divide, and are surprisingly widespread."

Draper's lab is concerned with two related questions about RNA. One involves the energetics of folding and the other asks how proteins recognize specific RNA sites and carry out specific tasks. 
Draper has relied on small angle X-ray scattering for information about RNA structure, but now has plans to collaborate with an expert in nuclear magnetic resonance (NMR) spectroscopy to help him further investigate riboswitches, to determine how they're folded up and where all the charges are.


The pursuit of biophysics requires the handling of an intimidating gauntlet of gadgets with names like excitation polarizer and automated titrating differential/ratio spectrofluorometer, most of which hum and wheeze and buzz behind locked and shrouded doors affixed with all manner of exclamatory signs: CAUTION! KEEP OUT! 

None conveys quite the gravity of the stuff housed in the NMR center, an underground sanctum occupying an unmarked space between Macaulay and Mudd halls at Homewood. There, three huge chrome canisters—think giant thermoses on steroids—have stairways and decking built around them for access. All stand on pneumatic legs designed to absorb vibrations. Deep inside of each, buffered by a layer of liquid helium to maintain very low temperatures, are big, powerful magnets.

The two large machines cost about $1 million each, and the one behemoth runs $3 million. PMB director Juliette Lecomte, a bespectacled biophysicist with a lilting Belgian accent, says her research involving hemoglobin proteins depends almost entirely on this powerful but complicated form of spectroscopy.

"It gives me access to information that I couldn't get any other way about the individual carbon, nitrogen and hydrogen atoms in my protein molecule," Lecomte says.

NMR spectroscopy produces more than pretty pictures of molecules. It detects when parts of the structures hiccup, fluctuate, and burp. It tells which regions of molecules are the softest, weakest, and strongest, and reveals potential breaking points—all vital details informing the deep understanding that's necessary whether one's aim is to design and construct new biological systems or fix broken ones.

Lecomte sits at a computer in her office and shows the kind of data that NMR experiments yield.

"Here, we're looking at signals—represented by crowds of dots—associated with each nitrogen-hydrogen unit in the protein," she says. "It's a beautiful mess of spots!" The spots are distributed over a wide range of frequencies, which means this protein is well-behaved, she says: "It's folded in a specific shape and gives me signals that, when I change the conditions, are going to respond and tell me something about the environment of the nuclei."

Suddenly it's clear: These spots are beautiful to her for their potential to be utterly revealing. Earlier, when we walked through her lab, Lecomte paused at the refrigerator, unable to resist rearranging a few words of magnetic poetry: "tell her all."

The meaning of her mini-haiku was delightfully ambiguous: Was it an entreaty to the paradoxical hemoglobin to reveal their secrets to her, or a prompt to her soft-spoken self to be candid with me?

Banking on the latter, I redirect a hard squint from the computer screen full of spots toward a Jackson Pollock painting hanging on her office wall.

"So, do you see any protein folding going on in that?"
 "Sometimes yes," she says, gazing at the gray-and-black spattered image. "Depending on how long I've been here."



Doug Barrick shows off a picture of a protein designed by his lab, where six graduate students and a research associate are working on several related projects. "It's based on nature," he says. "But it's better."

Better than nature?

"Yeah. Way better." I'm thinking this Birkenstocked biophysics prof has a mutated humility gene—perhaps attributable to the University Alumni Association Excellence in Teaching Award he received in 2008, or maybe because his lab's current bio-waste research has garnered coveted support from the provost.

But my hypothesis pops like a soap bubble when Barrick divulges his dishwashing roots in order to analogize his latest research. It's plain this teacher will go to great lengths to explain a complicated point in a way that anyone can understand.

Flash back a few decades: Barrick is a teenager, growing up in Boulder, Colo. After school, he washes pots and makes noodles at an Italian restaurant owned by his wrestling coach.

"It's boring, after a while, to cut pasta for 200 people a night," Barrick recalls. "So you start wondering, what else can I do with this stuff? How thick can I make it? How thin? How long?"

That last question prompted Barrick and his kitchen mates to roll and splice and roll and splice until they produced a fettuccine fit for the record books.

"We made the longest noodle ever," Barrick says. "We needed a measure of length that was pretty big, so we wrapped it around the dumpster a bunch of times."

A bunch? More quantitative please …

"Like five."

He says one of the projects going on now in his lab is similar, in a sense, to this adolescent prank.

"These repeat proteins we're making—the reason I can say they are better than any in nature is their remarkable stability," he explains. "They ain't never unfolding.

"That kind of stability makes them very useful for protein engineering. Right now, we're using repeat proteins as super-resistant scaffolds to which we can affix enzyme domains in particular orientations so they're reactive for specific purposes."

Barrick's team wants to design protein arrays for digesting waste biomass—the kind of stuff that fills dumpsters. Their goal, he says, is to build molecules that will cheaply and reliably break down the estimated one billion tons of waste biomass produced annually in the U.S. and turn it into simple sugars that can be used to make ethanols and other liquid hydrocarbon fuels.

"These repeat proteins get more stable the longer you make them," Barrick explains. "Varying their lengths has allowed us to learn a lot about the physical chemistry of how they fold. So far, we've strung 10 of these repeat protein units together. I've been bugging one of my students to see how big can we make the thing. He had what he needed to make repeats of 16 and 32, and he's going to try for 64 and 128. These would then be the long-noodle equivalent of this protein!"


As good as the PMB is, it might even be better if it were bigger, says Jon Lorsch, whose unfortunate task is turning away qualified candidates for a lack of available student slots. Despite meriting what may be the healthiest NIH training grant in the country, this program lacks the additional institutional funding necessary to fuel its growth as the field burgeons, Lorsch adds.

In fact, if the scientific disciplines that constitute biophysics continue to evolve—with all the traditional distinctions getting blurrier by the day—Garcia-Moreno predicts that within 100 years, "we're all going to be biophysicists."

 A stunning case in point is Jie Xiao, a biophysics professor in the School of Medicine to whom Garcia-Moreno refers as "the young generation," and whose work Juliette Lecomte sums up as: "Wow!"
In 2001, Xiao was studying biochemistry as a grad student when she heard about an emerging field called single-molecule biophysics. At the time, the business-as-usual methodology of studying millions of molecules all together in a test tube wasn't working particularly well for her. Instead, she was fascinated by the possibility of isolating single molecules of interest and watching how each carries out important biological functions. It was then that a Harvard biophysicist posed a spectacular question that, in an instant, changed her life's path: "Do you want to look at gene expression in real time?"

Duh. Does a molecule have atoms? She promptly became the very first biology postdoctoral fellow in her advisor's hard-core biophysics lab and set out to do single-molecule work in a way that had never been done before.

Even in a cutting-edge field, Xiao stands out as one of the cutting-edgiest. She's different from "usual" single-molecule biophysicists, as if such brilliant beings ever could be construed as pedestrian in any way. Instead of watching individual purified DNAs and proteins in a well-controlled man-made environment, Xiao moves single molecules back into living cells and studies them in the most complicated but natural physiological contexts. By focusing on one molecule at a time under a microscope, she's trying to make molecular movies of how these molecules work individually and collectively inside a living cell.

Xiao's arrival at Hopkins marked the PMB's foray into this forward-thinking area. Using complicated microscope-and-laser-system set-ups, she and her students manipulate single molecules in living cells to investigate the molecular mechanisms of both gene regulation and cell division. Her 5-year-old son, Leo, aptly dubbed her lab "the light saber room."

Which brings us full circle, to the point where we can finally pin down, once and for all, a universally agreed-upon definition of biophysics: It's science, not fiction.



How a handful of traditionally disparate fields at Hopkins have folded together to give shape to a discipline that, appropriately enough, creates order out of chaos.





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