Michael A. Raftery (1936–2007)—the first enzyme mechanism, sequential cooperativity, and the nicotinic acetylcholine receptor defined

Michael A. Raftery, brilliant scientist and eclectic wit, left us on August 1, 2007, after a battle with cancer. He was with his family in Chico, California: his former wife, Judith H. Raftery, and his dearly loved children: M. Daniel Raftery and his family, Jennifer R. Raftery, and John K. Raftery and his wife. It meant a great deal to Mike that he lived to enjoy John's wedding in Chico on June 2, 2007.

Michael Raftery developed innovative experimental approaches in multiple areas of emerging biochemistry. He conducted seminal experiments defining the catalytic pathway of the first enzyme whose structure was known to atomic resolution. He demonstrated that the cooperativity exhibited by hemoglobin, the paradigmatic oligomeric protein of the day, is not as simple as originally proposed. His research revealed the molecular makeup and ion-flux mechanism of the first neurochemical receptor to be isolated.

Michael grew up in Mayo, Ireland. He was a chemist of extraordinary ability, an athlete, and a sprinter in his undergraduate years. He earned a B.Sc. degree in chemistry in 1956, and a Ph.D. degree in biochemistry in 1959 at the National University of Ireland in Galway. From 1960 to 1964, he worked at the University of California in Berkeley doing postdoctoral research as a Joseph P. Kennedy, Jr. Foundation Fellow, first with Professor Choh Hao Li and then Professor R. David Cole. There he pioneered the aminoethylation of cysteine residues as a means of generating new tryptic cleavage sites, useful in sequencing proteins long before the advent of gene cloning or mass spectrometric sequencing. In Berkeley, he met and married Judith H. Rosenberg. They had three children—Daniel (now a chemistry professor at Purdue), Jennifer (now a prop manager in Hollywood), and John (now a loan officer in Chico, California).

Michael Raftery obtained his first faculty position as the Arthur Amos Noyes Instructor at the California Institute of Technology in 1964. There he took on the mechanism of lysozyme, the first enzyme for which the three-dimensional structure had just been determined. He and his Ph.D. students, Frederick Dahlquist (now professor at UC Santa Barbara) and Stanley Parsons (now professor at UC Santa Barbara), had a great impact on enzymology and protein chemistry, first harnessing NMR spectroscopy among other methods to establish the manner in which substrates bind to lysozyme. In approximately 20 fine publications, they provided evidence that the chemical reaction proceeds through an oxocarbenium ion-like transition state whose formation is catalyzed by glutamic and aspartic acid residues of suitably altered pK _a values. Raftery and his Ph.D. student Wray Huestis (now professor at Stanford) also made key contributions to understanding oxygen binding by hemoglobin, whose structure was approaching atomic resolution. Raftery and his group used NMR to prove that oxygenation produces sequential changes in the conformations of the four subunits. These observations are a landmark in the history of protein cooperativity.

Raftery was among the first biochemists to recognize how the then emerging, three-dimensional structures of proteins could instruct experimental determination of functional mechanisms. His wily intuition translated the structures of lysozyme, other enzymes including glyceraldehyde-3-phosphate dehydrogenase, and hemoglobin into detailed chemical hypotheses and experiments that led to biochemical mechanisms that appear in textbooks today.

Raftery was appointed associate professor (1969) and professor (1972–1987) at Caltech. By 1970, Raftery brought his chemical insights to bear on fundamental questions in the mechanisms of neuronal signaling. Already in the 1930s, following observations of Otto Loewi and H.H. Dale that acetylcholine acts as a neurotransmitter at the neuromuscular junction, David Nachmansohn had shown that the electrically excitable organs of electric fish can process vast amounts of acetylcholine. In their own careers, Jean Pierre Changeux (soon to move to the Institut Pasteur) and Arthur Karlin, both at the Columbia School of Physicians and Surgeons (to which Nachmansohn had moved in 1942), and Michael Raftery took advantage of the electric ray Torpedo californica and the electric eel Electrophorus electricus as rich sources of neurochemical elements. These species harness stacks of aligned cells containing large amounts of the nicotinic acetylcholine receptor or the voltage gated sodium channel to generate charge flow. Raftery showed that the receptors can be extracted from membranes using detergent and then purified using their affinity for neurotoxins. In 1974, he showed that purified receptors can be reconstituted in active form into artificial liposomes, thus setting the groundwork for extensive in vitro characterization of this fundamental paradigm of neuronal signaling.

In one of many highly productive years, of the 25 papers he published in 1979, Raftery and his group produced 10 back-to-back publications in a single issue of the ACS journal Biochemistry (1979, volume 18, issue 10), of which nine described the acetylcholine receptor, and one described the sodium channel. In that issue of Biochemistry, Raftery showed that the nicotinic receptor is composed of four subunit types that are variously glycosylated and phosphorylated. He separated the subunits from other associated intracellular proteins, notably from the 43-kDa protein that he showed is not a part of the functional receptor. He developed fluorescent labels and elegantly used them to identify agents that evoke conformational changes associated with opening of the channel and desensitization, the phenomenon in which receptors lose sensitivity to ligands after extensive stimulation. He defined different types of ligand binding sites and their actions, a topic that continued to fascinate him until his passing. He described cation conductance properties and showed that neurotoxins compete with acetylcholine for certain ligand sites on the receptor. By 1979, Raftery and Changeux described sequences of the first 25 and 20 amino acids of the α chain, respectively. In a landmark paper published in Science in 1980, Raftery and colleagues determined the amino acid sequence of the first 56 residues of each of the four subunits. They showed that the sequences of all four subunits are similar, being evolutionarily diverged from a common ancestral subunit.

Beginning a long collaboration with Michael Raftery, my own group produced the first three-dimensional structure, and image reconstructions of the structure and organization of the receptor in membranes. The images and similarity of subunit sequences immediately led to the conclusion that a quasi-fivefold symmetric pentamer of subunits surrounds a central ion-conducting channel. We also deduced the arrangement α-x-α-y-z- for the subunits (in parallel with Karlin's determination of β adjacent to Δ), predicted the topography of chains and their secondary structures, and sequenced the oligosaccharide groups.

Raftery provided the amino acid sequences of all the subunits at a crucial time, when DNA technology had to rely on probes derived from the protein sequence. He enabled the emerging field of cloning and determination of complete nucleic acid sequences specifying the subunits and their diversity. Because of Raftery's discovery, in Japan, Shosaku Numa was able to develop probes for all the receptor genes, and the probes, in turn, showed that different isoforms of each subunit type exist. Thus, as many as 10 genes for α subunits, four for β subunits, and so on, allow for great diversity in the types of nicotinic receptors that are assembled in different parts of the nervous system. Raftery's preparation of pure membrane protein subunits and their N-terminal sequencing in 1979/1980 were revolutionary in their time, and serve as a defining landmark that enabled so much of what followed in the field.

In 1971, Michael was awarded the Doctor of Science degree from the National University of Ireland in recognition of his outstanding research contributions; in 1986, he was elected a fellow of the Royal Society (FRS), the highest UK honor granted to an Irish scientist; and in 1987, awarded the Doctor of Laws degree from the National University of Ireland.

Michael Raftery and I continued our collaboration over 36 years to purify and crystallize the nicotinic acetylcholine receptor for determination of its structure. With Michael Shuster and Julian Chen in my group, we succeeded with receptors from three species, although not yet with adequate resolution to probe the mechanism of gating.

In 1987, Michael moved from Caltech to the University of Minnesota. He continued his collaboration with Susan Dunn (now professor at the University of Alberta) to pursue their observation that the binding sites that open the channel have much lower affinity for acetylcholine than those that desensitize the channel. Dunn and Raftery (2000) demonstrated that occupancy of the high-affinity sites with a moderate concentration of agonist does not desensitize the receptor toward subsequent application of a high concentration of agonist. The observation implies that high-affinity sites are not directly involved in activation, and occupancy of these sites alone does not induce receptor desensitization. This interpretation remains underappreciated, although it makes abject sense. Upon being engulfed by an initial wave of ∼1 mM acetylcholine that is “puffed” into the synapse by the nerve terminal, the receptor rapidly opens its ion channel. As the concentration of acetylcholine rapidly falls because of hydrolysis by acetylcholine esterase in the synaptic gap, the ion channel closes in ∼1 msec without desensitization. The phenomenon of desensitization may have no normal physiological significance, Raftery argued, and the high-affinity binding sites that most other researchers monitored do not open the channel. The most recent publication by Raftery, Dunn, and collaborators appeared in February 2008.

Michael Raftery became emeritus professor at the University of Minnesota in 2001 and returned to live in Berkeley, the town of his postdoctoral work and marriage to Judith. Throughout his scientific life, Michael recognized key questions in biochemistry and searched for new techniques, often provided by chemical thinking, which could be brought to bear on answering those questions. For example, he recognized the potential usefulness of aziridines and oxonium salts to aqueous protein chemistry, NMR spectroscopy to protein biophysics, and kinetic isotope effects to enzymology. He published many of the first papers in biochemistry and biophysics that used these techniques. Michael also published on the vitelline envelope of eggs, half-site reactivity in oligomeric proteins, lipid biophysics, and isolation of hyaluronidase, among other topics.

For Michael, science was intense, creative, and highly competitive. There had to be solid data that led to unequivocal conclusions likely to withstand the test of time. Observations that led to conjecture he challenged vociferously. He was a scientist of the highest caliber.

Michael Raftery was endearing company at any gathering of people, always seeing and enjoying the humorous or incongruous side of events, daily encounters, literature, politics, religion, and world news. He possessed a keen command of the English language and always brought laughter and enjoyment, mirth and happiness to the company around him with his incisive wit.

Michael was also a spectacular chef. One time he planned to prepare beef Wellington for a special dinner. As Judy recalls, he tried the dish ahead of time, didn't like the result, found a better recipe, tried that recipe also ahead of time, and finally created a memorable Wellington on the projected day. My family and I saw him many times for dinner, celebrations, holidays, and conversation. When serving as host and cook at a celebration, he often would decorate a cake with memorable finesse and art. When dining in a restaurant, he would hold the waiters to the same high standards. He would expect them to inform on the nuances of the cuisine, suffering no lack of detail in the methods of preparation and origins of ingredients. This interrogation often led to minor embarrassment as questions reached the limits of the waiter's knowledge. Pretension was not for Michael in any sense, whether it be in science, culinary arts, literary wit, or life.

Michael Raftery left us too soon, but he left us with fond memories and important lessons. Never compromise in science or life, as the true answer is the only one that will stay, and he left many of us with his absolute loyalty, wonderful taste in humor and food, and the finest sense of what constitutes excellence. We mourn his loss and remember the light he brought to us all.

ROBERT M. STROUD
Biochemistry Department, University of California, San Francisco, California