SCIENCE & TECHNOLOGY tells C&EN. It wasn't until he enrolled at nearby Brandeis University that "my eyes were opened to the idea that, as a scientist, I could spend my life as an explorer." He chalks this lesson up to his undergraduate research mentor, Brandeis biochemistry professor and ion channel enthusiast Christopher Miller. But despite AMANDA YARNELL, C&EN WASHINGTON Miller's urging that MacKinnon go to graduate school, MacKinnon opted for SK PETER C. AGRE OF J O H N S MacKinnon grew up in Burlington, medical school after obtaining his B.A. in Hopkins University School of Mass. As a child, his scientific interests in- biochemistry from Brandeis. It wasn't unMedicine if he considers him- cluded examining bugs and blades ofgrass til after he received his medical degree and self a chemist, and he'll tell you under a microscope. "But I wasn't a great completed his residency that "it finally that he prefers the term "re- student. I was kind of a dreamy kid," he dawned on me that I would really rather be doing the kind ofproblem solving that naissance man." 'Vet Agre, a physician you can do in a laboratory," MacKinspecializing in blood disorders who non says. turned his full attention to research only midway through his career, shares this So after his residency he returned to year's Nobel Prize in Chemistry with Miller's lab at Brandeis to learn the another physician-turned-bench-scienropes as apostdoc.There, MacKinnon tist, Rockefeller University's Roderick studied potassium ion channels, memMacKinnon (C&EN, Oct. 13, page 11). brane-spanning proteins that are cruAlthough neither Agre nor Maccial for nerve function and the beating Kinnon readily identifies himself as a ofyour heart. MacKinnon puzzled over chemist, their work defining how ions how these proteins transport K+ ions and water move in and out of cells "is but block passage of Na+ ions, which chemistry at its best," according to are only slightly smaller. Moreover, he Gregory A. Petsko, a professor of wondered, how do they maintain this chemistry and biochemistry at Branexquisite selectivity while moving deis University potassium ions through the channel at a rate of 108 ions per second? As protection from their environment—and to keep their proteins, WHEN HE STARTED his own lab at RNA, and DNA from escaping—cells are enclosed in a membrane made of a Harvard University MacKinnon spent double layer oflipids. But cells also need years trying to answer these questions a way to move ions, water, and other INSIGHTFUL Agre shares this year's by painstakingly mutating individual molecules across this waxy barrier. The chemistry Nobel Prize for discovering a class amino acids in potassium ion channels solution: membrane-spanning proteins of cellular water channels called aquaporins. and then testing how each mutation afthat act as pumps or channels. Jens C. fects channel function. "But it became Skou, a physician and biophysicist, clear that I would never understand it shared the 1997 Nobel Prize in Chemwithout seeing the channel," Macistry for discovering the first membrane Kinnon says. "That's what drove me protein pump. This year, membrane to X-ray crystallography" channels got the nod. Membrane proteins are notoriously difficult to crystallize. At the time, most scientists thought that obtaining AGRE, 54, took home half of the $1.3 a 3-D structure ofan ion channel would million prize for discovering thefirstof be extremely difficult, if not impossia class of membrane-spanning proteins ble. But that didn't deter MacKinnon. that allow cells to regulate their volAgainst his colleagues' advice, he abanume. These proteins, which Agre doned his existingprojects and plunged named aquaporins, provide a pathway into X-ray crystallography, a technique that allows water—but not ions or othabout which he knew nothing. er small molecules—to rapidly travel across cell membranes. The other half "I got it in my head that I wanted of the prize went to MacKinnon, 47, a to go for broke," MacKinnon says. He Howard Hughes Medical Institute insat in on an undergraduate course on vestigator who transformed the field the subject and took to lurking in the of ion channels just dye years ago when lab of an established X-ray crystalloghe published the first high-resolution rapher at Harvard. In 1996, when structure of an ion channel—in this FEARLESS MacKinnon was honored for his Rockefeller University offered him a case, one that lets potassium ions structural and mechanistic studies of job and promised to fund his risky projthrough but nothing else. ect, only one member ofMacKinnon's potassium ion channels.
RENAISSANCE MEN Chemistry Nobelists solved basic problems about how ions and water cross cell membranes
A
HTTP://WWW.CEN-ONLINE.ORG
C & E N / N O V E M B E R 3, 2003
35
SCIENCE & TECHNOLOGY 100
YEARS
AGO
Arrhenius' Theory Of Electrolytic Dissociation, Once Dismissed, Won Him 1903 Chemistry Nobel
I
t's difficult to imagine that one of the most commonplace ideas reach far and deep. Though his work on ionic dissociation won him in all of chemistry—that ionic and polar covalent compounds the most accolades, chemistry students perhaps recognize him for separate into oppositely charged ions in solution—was the famous equation that bears his name. The equation relates once heretical. temperature to reaction rates. When Svante A. Arrhenius proposed the concept Arrhenius also developed the concept of activation of electrolytic dissociation in his physics Ph.D. energy—the energy required to initiate a chemical re dissertation at the University of Uppsala in action—another key variable in the Arrhenius equa 1884, his advisers were not impressed. True, tion. And he introduced the definition of acids as Arrhenius' theory neatly explained why salt producers of hydrogen ions in solution and of and water individually don't conduct electrici bases as producers of hydroxide ions in solution. ty well, yet a salt solution does. Later on in his career, Arrhenius applied his But most scientists at the time dismissed theories to immunology, coining the word "imthe idea that in water, sodium chloride sepa munochemistry." He predicted the greenhouse rates into charged versions of its components, effect, an atmospheric warming caused by an im sodium and chlorine. After all, the two ele balance of C02. And he hypothesized that life on ments are highly reactive by themselves: Sodi Earth was seeded by spores from other worlds, um bursts into flame when it touches water, and driven here by radiation pressure in space. chlorine is a poisonous gas. Arrhenius' advisers The Royal Swedish Academy of Sciences' biography sent him off with the lowest possible passing rank. of Arrhenius describes him as "a contented man, happy in his work and in his family life." He was married twice, first It was years later, after the staunch support of a COURTESY OF ROYAL SWEDISH ACADEMY in 1894, to Sofia Rudbeck, one of his pupils. They had one number of chemists, notably that of Wilhelm Ostwald OF SCIENCE son and divorced two years later. In 1905, he married and the increasing weight of experimental evidence, Maria Johansson; they had three more children. that the idea won acceptance. And in 1903, the Nobel committee vindicated Arrhenius with a Nobel Prize in Chemistry. Arrhenius was a physics professor at the Royal Institute of Technology of Stockholm in the late 1890s. In 1905, the Swedish Arrhenius, born in 1856 in Sweden, is considered one of the Academy formed the Nobel Institute for Physical Chemistry and founding fathers of physical chemistry, along with Jacobus H. made Arrhenius its director. van't Hoff and Ostwald. Van't Hoff won the first Chemistry Nobel Prize in 1901 for his work on solutions, and Ostwald was award In 1927, Arrhenius died at age 68. Some speculate that his ed the Chemistry Nobel in 1909. death was due, in part, to the excess work his scientific populari Arrhenius' contributions to fundamental general chemistry ty demanded.-ELIZABETH WILSON
lab chose to come along. MacKinnon's a narrow, mostly hydrophobic channel to a wife, Alice, who has a bachelor's degree in large water-filled cavity in the membrane's chemistry from Brandeis, worked with center. There, they are stabilized by partial them, "I think because she felt sorry for negative charges provided by the dipole me," MacKinnon says. moments of short α-helices until passed to MacKinnon's bold gamble paid off in the "selectivityfilter,"a line ofpotassium ion 1998. That year, his lab published the first binding sites created by the backbone carhigh-resolution structure of an ion chan bonyl oxygens of a string of highly con nel [Science, 280, 69 (1998)}. Their 3.2-A served amino acids. Ions move from one structure of the potassium channel pro site to the next like a bucket brigade. tein KcsA from Streptomyces lividans—in which the channel's four identical subunits EACH OF THESE binding sites closely form an inverted teepee—rocked the ion mimics potassium ions' octahedral hydra channel field. tion shell, thereby minimizing the energy Subsequent higher resolution structures required to strip off their water coats. Be from MacKinnon's lab revealed the mech cause of their smaller size, sodium ions anism of action and selectivity of potassi don'tfitin these binding sites as snugly and um ion channels infinedetail. These struc thusfindthe energetic cost of trading their tures have shown that ions, still bearing water coat for a spot in the selectivity fil their coat ofwater molecules, travel through ter too high. Recently, MacKinnon has col
laborated with Rockefeller professor Tom W Muir to synthesize channels containing chemically modified selectivity filters. "This will allow us to explore the contri butions of the K+-coordinating backbone oxygens in a way impossible by conven tional mutagenesis," MacKinnon notes. MacKinnon has gone on to unlock the mysteries of how these channels open and close in response to external cues. His most recent structure, of a potassium channel that opens in response to voltage differ ences across cell membranes, has challenged the widely held model of how these channels work[Nature, 423,33 and 42 (2003)}. "The movement of ions across mem branes is a problem that has interested chemists for decades," notes biochemistry professor Richard N. Armstrong of Vanderbilt University "The real value of Mac-
The movement of ions across membranes is a problem that has interested chemists for decades." 36
C&EN / NOVEMBER
3,
2003
HTTP://WWW.CEN-ONLINE.ORG
Kinnon's work is that it shows one way nature solved the problem of transferring ions in a controlled and specific fashion." Although MacKinnon's path to discovery was carefully plotted, Agre's was not. A practicing hematologist atJohns Hopkins, Agre was trying to purify Rh factor— a protein found on the surface of red blood cells that defines whether one's blood type is positive or negative—when he stumbled upon the water channel protein that won him the Nobel Prize. Agre, who grew up near MinneapolisSt. Paul in Minnesota, willingly admits that his early trajectory was less than Nobelworthy: He withdrew from high school halfway through his senior year. "At the time, I was getting a D in chemistry," Agre recalls. "Ifmy high school chemistry teacher is still with us, he probably aspirated his morning coffee when he heard the news." This early stumble dismayed his father, Courtland Agre, who was a professor of chemistry, first at St. Olaf College and then at Augsburg College. The elder Agre tried to share his love of chemistry with his six children, "but like other animal instincts, whatever your parents think is cool is uncool," Agre says. As a ninth grader, Agre had the chance to meet chemist and twotime Nobelist Linus Pauling, whom the elder Agre had invited to speak at Augsburg and spend the night at the Agre home. Peter Agre found Pauling "inspiring," but the event failed to convince him to take up chemistry as a career. His father's influence did inspire his choice of major, however. Agre studied chemistry at Augsburg, then went on to earn his medical degree in 1974 fromjohns Hopkins, where he eventually returned in 1981. The protein contaminant that frustrated Agre's attempts to purify Rh factor is remarkably abundant in red blood cells— each cell has about 200,000 copies. Figuring that anything that abundant was likely to be important, Agre tracked down the gene that encoded the protein and realized that the protein was present in kidney cells and plant cells, too. On a tip from a colleague, Agre began to consider whether it might be a water channel. At that time, many scientists thought that water passed into and out of cells by simply diffusing through the membrane. As early as the 1950s, biophysicists had demonstrated that in some cells, water got in and out at rates much faster than could be explained by simple diffusion. They suggested that these cells might contain water-specific channels. But this proposal remained controversial, largely because no one had been able to find a water channel protein. HTTP://WWW.CEN-ONLINE.ORG
Agre's landmark experiment neatly cleared up the controversy [Science, 256, 385 (1992)}. Agre expressed the mysterious protein in frog eggs, which are normally impermeable to water. When these eggs were transferred to freshwater, water quickly rushed in and "they popped like popcorn," Agre tells C&EN. "It was immediately clear our contaminant was a water channel." Agre's identification of the so-called aquaporins has also paved the way for investigation of their structure and function. In collaboration with biophysicist Yoshinori Fujiyoshi of Kyoto University, inJapan, and structural biologist Andreas Engle of the University of Basel, in Switzerland, Agre used electron microscopy to obtain a 6-Â structure of human aquaporin 1 [Nature, 387,624 (1997)}. The structure revealed a tetrameric, hourglass-shaped protein containing four pores, each just large enough for water to pass through in single file. Still, at this resolution, Agre's team could not make out the locations of individual amino acid side chains. So just how each pore manages to rapidly transport water—yet nothing else—remained murky until higher resolution structures became available, including an improved structure from Agre's team, as well as one by structural biologist Robert M. Stroud of the University of California, San Francisco, of aglycerol-transporting aquaporin relative from bacteria [Science, 290,481 (2000)} and another of aquaporin 1 purified from a cow [Nature, 414, 872 (2001)} by structural biologist Bing K. Jap of Lawrence Berkeley National Laboratory
These high-resolution structures re vealed the inner workings of aquaporins in atomic detail. Water molecules travel sin gle file through a narrow pore that con nects water-filled intracellular and extra cellular vestibules. This 28-Â-long pore is lined with a row of eight carbonyl oxygen atoms that can accept hydrogen bonds from the queue of water molecules, "ensuring that every water molecule is precisely oriented throughout the channel," Stroud says. But because the rest of the pore is lined with mostly hydrophobic amino acids, "water-pore interactions are kept to a minimum," Jap says, allowing water to rush through each pore at a rate of 3 x 10 9 molecules per second. FOR A SHORT STRETCH, the pore narrows to just wide enough for a water molecule to pass, preventing larger molecules from entering. And ions—which would have to abandon their bulky water coats to enter—don't do so because the pore isn't a good substitute. In addition, a conserved, positively charged arginine residue in this region prevents cations, including protonated water (H 3 0 + ), from entering. Nature has gone to great effort to prevent protons from passing through, too. Previous structural work had suggested that a pair of asparagine residues as well as the dipole moments of short α-helices would force water molecules to flip near the pore's center. This forced reorienta tion—which was confirmed by molecular dynamics simulations performed by teams led by biophysicists K. Helmut Grubmuller of the Max Planck Institute for Bio-
•\Vt
ψ%\^^ f
S
t
-%-4fc
^
CRYSTAL CLEAR At left, the protein backbones of two subunits of the KcsA potassium channel are shown with its cytoplasmic gate closed. Four potassium ions bind at four sites in the selectivity filter (yellow). Another potassium ion waits its turn in the water-filled cavity, still wearing its coat of waters (shown as red dots with blue outlines), while two others are seen on the extracellular side of the membrane. A close-up of the selectivity filter is pictured on the right (yellow = carbon, red = oxygen, blue = nitrogen, green = potassium).
C&EN
/ NOVEMBER
3, 2 0 0 3
37
SCIENCE & TECHNOLOGY
LINEUP At left, the protein backbone of one subunit of bovine aquaporin 1 is shown as a ribbon, with the dimensions of the water pore shown in blue dots. At right, water molecules (space-filling models) move through the pore single file. Residues defining the constriction region are shown as ball-andstick representations. In particular, a histidine protruding into the pore ensures that molecules larger than water can't enter. (Gray = carbon, blue = nitrogen, red = oxygen.)
physical Chemistry and Klaus Schulten of the University of Illinois, Urbana-Champaign—breaks the continuous chain of hydrogen-bonded waters and prevents protons from "hopping" along the hydrogenbond network. Stroud tells C&EN that his lab will soon publish the structure of a bacterial aquaporin in the recently launched online jour-
cargo rapidly and selectively The crucial roles these membrane channels play in physiology suggests that such knowledge may someday lead to treatments of a variety of ailments. In addition to playing an important role in water uptake by plants, aquaporins may play a role in some kidney conditions and brain swelling after strokes. Agre and others have shown that amino acid mutations in these water channels are linked to a number of human diseases, including one that causes cataracts in children and one that is characterized by an inability to reabsorb water during urine production. And abnormal expression of aquaporins may lead tofluidoverload during pregnancy and congestive heart failure, he tells C&EN. Ion channels are known to play a role in cardiovascular disease, hypertension, epilepsy, and a number of neurological disorders. "The pharmaceutical industry is very interested in ion channels,,, MacKinnon tells C&EN, noting that many drugs already in use target such proteins. Although new drugs inspired by his structures are far off, he notes, "our structures have planted the seed for ways that we might be able to manipulate ion channels with small molecules." •
nal PloS Biology. Unlike purified human and bovine aquaporins, this bacterial aquaMORE ONLINE porin can be used for mutagenesis studies, Animations of the movement of water Stroud says, allowing the contributions of through aquaporins, as well as Agre's and individual amino acids to be parsed out. MacKinnon's first-person tales of the "Aha! Over the past decade, Agre and MacMoments" that led to their Nobel Prize, can Kinnon have made breathtaking strides in be found on C&EN Online, http://www. the understanding of how potassium ion cen-online.org. channels and aquaporins transport their
Polymer Enhancement Commercial Quantities Available
THPE-GE is a reactive intermediate targeting high temperature adhesive and structural resin applications. This strong cross-linking or branching agent can enhance key properties and improve product performance. Suitable for castings, laminates, composites and coatings requiring high mechanical strength, heat or chemical resistance.
Adhesion
THPE-GE C T ^ " l,l,l-tris(p-Hydroxyphenyl)ethane Glycidyl Ether The DuPont Oval logo, DuPont™, The miracles of science™, are trademarks or
Hardness
For more information, call Matt Romberger, at 972 720-5016, or visit www.cfem.com. Formerly, TriQuest, LP
Heat resistance
Solvent resistance
DuPont Electronic Technologies JPQÎH? The miracles of science*
d trademarks of E.I. du Pont de Nemours and Company.
38
C&EN
/
NOVEMBER
3.
2003
HTTP://WWW.CEN-ONLINE.ORG
