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In memoriam: Reflections on Fred Richards (1925–2009)

2009; Wiley; Volume: 18; Issue: 4 Linguagem: Inglês

10.1002/pro.81

1469-896X

Robert L. Baldwin,

Digestive system and related health

Fred Richards made a remarkable discovery right at the start of his scientific career. In his year of postdoctoral research (1954–1955) at the Carlsberg Laboratory, Copenhagen, Fred studied the digestion of RNase A (bovine pancreatic ribonuclease A) by subtilisin, a bacterial protease. He found evidence for a surprising intermediate in the digestion process: see below. Later, in his own lab at Yale, Fred characterized the intermediate (RNase S) by separating it into two parts, a 20-residue peptide (S-pep) and a 104-residue protein moiety (S-prot), and he found that enzymatic activity is lost when the two chains are separated. Remarkably, S-pep and S-prot combine spontaneously when they are mixed together and the product is active enzyme. This result1 immediately clarified how peptide hormones might activate their protein receptors. Because S-prot is only weakly folded and S-pep is nearly structureless, whereas RNase S is a stable, well-folded protein, the reaction S-pep + S-prot = RNase S became a standard system for folding studies and helped to open up the study of the mechanism of protein folding. At that time, Linderstrøm-Lang's lab was a world center for studying protein folding but those were early days and people were still searching for tools to study folding and for insights that would open up the folding problem. Lang himself (as the lab members usually spoke of him) was developing the new tool of hydrogen-deuterium exchange between peptide NH protons and solvent (D2O), and he had shown that the H-D exchange rate is exceedingly sensitive to the extent of folding. Walter Kauzmann was a sabbatical visitor during this time and his laboratory at Princeton was proving that protein denaturation is synonymous with unfolding the three-dimensional structure. Chris Anfinsen was making one of his frequent visits. Three years later he would light up the protein folding field by showing that that the free energy change upon folding drives the folding process. John Schellman, then doing postdoctoral research, was analyzing theoretically how much peptide hydrogen bonds contribute to the stability of an α-helix or β-sheet in water. Other notable visitors to the Carlsberg lab in 1954–1955 included Charlotte Green (John Schellman's wife-to-be), Bill Harrington and Harold Scheraga. Fred himself wrote a vivid description of the scene in the Carlsberg lab in this period.2 The level of scientific excitement was high and Linderstrøm-Lang's love of life, and especially music, spilled over and set the atmosphere. Fred was given the subtilisin digestion of RNase A to study because earlier Martin Ottesen and Lang had found that subtilisin converts ovalbumin to a new protein form (plakalbumin) when a small peptide is released.3 Surprisingly, plakalbumin still crystallizes although the crystals are now thin plates, a different crystal form than that of ovalbumin itself. Lang wanted to know how subtilisin might change the properties of other well-studied proteins; there were only a few such proteins at that time. Fred's first paper on the digestion of RNase A by subtilisin continues where Sumner Kalman (an earlier Carlsberg postdoc) left off.4 The initial digestion results were not easy to interpret but they did contain the suggestion that there is an enzymatically active intermediate. In most proteolytic digestion experiments of that period, only small peptide fragments were found. In his Carlsberg recollections,2 Fred pokes fun at the Kalman et al. paper: see legend to Figure 5 of Ref. 2 Before he left the Carlsberg lab, Fred had purified the enzymatically active intermediate but had not yet discovered its surprising properties5; these were revealed later in Fred's own lab at Yale. On the key paper reporting the separation and recombination of the two chains of RNase S,1 Fred was the sole author without help from students or postdocs. Fred never did this kind of work again, which required making sense of what first appeared to be a rather complicated biological system, but his 1958 paper demonstrated he could do it. While still at the Carlsberg Laboratory, Fred received an offer from Joseph Fruton in the Yale Medical School to join his Dept. of Biochemistry as an Assistant Professor. Fred accepted and moved to Yale. Three years later, the first x-ray structure of a protein, the 6-Å structure of myoglobin, appeared and Fred resolved to get the x-ray structure of RNase S. Fred's PhD research had prepared him for this: he worked with Barbara Low, an x-ray crystallographer in the Cohn and Edsall laboratory in the Harvard Medical School. For his thesis, he developed an accurate method for determining the molecular weight of a protein based on its unit cell dimensions and the density and composition of the crystal.6 From the group who had solved the myoglobin structure, Fred recruited Hal Wyckoff both to the RNase S project and to Yale. They published a medium-resolution x-ray structure of RNase S in 19677 and followed with a 2.0-Å resolution structure in 1970.8 RNase S was among the first half dozen or so proteins to have their x-ray structures determined at atomic resolution. After 8 years at Yale, Fred was recruited by President Kingman Brewster to build a new department. Fred had a major talent for recognizing exceptional ability in budding scientists and the department that emerged from his efforts was recognized worldwide. Initially, Brewster moved Fred from the Medical School to the north campus in 1963 and made him Chair of the existing Dept. of Biophysics, which had been slanted towards radiation biology. Reflecting its new direction, the department was renamed Molecular Biology and Biophysics. Later, after Fred's first term as Chair (1963–1967) and subsequent sabbatical, Brewster merged the new department with the Biochemistry Dept. in the Medical School, again enlisting Fred as Chair for a second term (1969–1973). The combined department was renamed Molecular Biophysics and Biochemistry (MB&B). Fred recruited six more faculty members at the junior level, including Don Engelman, Peter Moore, Tom and Joan Steitz, and Dave Ward. They joined Dieter Söll, Lubert Stryer, and other well-known scientists in the existing department. The seven of Fred's hires mentioned here would become members of the National Academy of Sciences. Fred encouraged the young people he hired to recommend others, and he identified at least two people in this way. Fred spent his sabbatical (1967–1968) in David Phillips' laboratory for structural biology at Oxford. It was the period when protein x-ray structures were still built by hand, initially from brass rod models, later from push-fit plastic parts. Because of the huge number of atoms involved and the precise alignments that were needed, building a protein structure was a complex undertaking. Fred lightened the task by devising a box containing a half-silvered mirror that allowed one to visualize the electron density map while building the structure.9 The apparatus was known colloquially as the Richards Box or Fred's Folly. People used it gratefully until computers took over the model-building process. On their way to the sabbatical in Oxford, Fred and his wife Sally sailed across the Atlantic to England in their own sailboat with just two crew, a notable sailing feat. Then Fred shipped the sailboat home. Fred undertook the development of a world-class center for x-ray structural work at Yale. He became the principal investigator of an NIH program project grant that included himself, Hal Wyckoff, Don Engelman, Peter Moore, and Tom Steitz (the WERMS group). They consolidated the x-ray and auxiliary equipment in a single core facility, available for use by everyone. Their center was extremely successful and Yale became the place to go if you had crystals of a protein and wanted to solve its x-ray structure. Fred could see far ahead both in his own research and in how an organization should develop. People recognized this ability and respected it. I remember particularly a Proteins Gordon Conference in 1973. Studying protein synthesis on polyribosomes was popular at that time and the eyes of the audience were beginning to glaze over from looking at too many sucrose gradient slides. When the moment came to choose the co-chairmen for the next conference, someone suggested abandoning the Proteins Conference and merging it with the Nucleic Acids Conference. With fire in his eyes, Fred stood up and foretold told a future when more protein structures had been determined: we would understand how proteins are folded, how they breathe and move, and how they interact with other proteins. Persuaded by this vision, the conferees voted for new chairmen who would point the field in the direction Fred outlined. Fred's prescient understanding of where protein research was headed and how organizations should develop led to many honors and responsibilities. He was elected President both of the Biophysical Society and the American Society of Biological Chemists (now ASBMB). When the Burroughs-Wellcome Foundation set up its interface program to bridge the gap between biology and mathematics and physics, Fred was asked to advise on the choice of members of the first Awards Committee and then to chair it. He had a broad knowledge of biophysics and knew who was doing exciting work. Fred was a member of the first Council of the Protein Society and when his term was finished, the Council sometimes asked Fred to serve on special committees with such charges as choosing the new Editor for Protein Science. Strikingly, Fred was made Chair of the special committee set up to advise the NIH Director on "the Gallo/Montagnier debacle" (Fred's phrase).10 A pointed description of his experience on that committee is given in Fred's autobiographical memoir11 entitled characteristically: "Whatever happened to the fun?" One of the committee tasks that Fred especially enjoyed was being a Director of the Jane Coffin Childs Fund (1976–1991). His job was to oversee the selection of postdoctoral fellows; the Fund had supported more than 800 fellows at the time of Fred's 1997 memoir. Fred could be refreshingly outspoken, as this anecdote from Mark Hermodson illustrates. "Fred was great on the Council in the early days of the Protein Society, not afraid to talk sense to strong characters.... I'll never forget his left-handed compliment to me after the first meeting I chaired in 1991. We were facing imminent bankruptcy and one council member was focused on starting the journal without any real concerns for the Society treasury. In addition, I had to tell another member to leave the room, due to a possible conflict of interest, and this made him mad. Afterwards, Fred said something like "Good meeting, I didn't think you could do it," which I understood was high praise." Another example of Fred's willingness to call a spade a spade is his description of E.J. Cohn2: "Cohn was a brilliant man, a first-class scientist, an excellent organizer and a major-league tyrant." Fred was determined to reserve some of his time for his own personal research. Tom Steitz describes Fred at work in the '70s. "At 4 PM sharp, he would leave his office and go to the computing center, which had an IBM 360.... He would... (spend) 1–2 h writing a program and resubmitting it for processing.... (After about) a year this gave his sole author paper on the packing density of proteins." One of Fred's major interests in the '70s was in the surfaces of proteins. The surface is where a protein interacts with ligands or other proteins or nucleic acids. Fred wanted an accurate physical method for describing the surfaces of molecules but he couldn't find such a tool in chemistry, so he and B.-K. Lee invented one.12 The Lee and Richards algorithm rapidly became a familiar tool to protein chemists. It employs a rolling sphere roughly the size of a water molecule (radius 1.4 Å) whose passage covers the entire protein molecule and gives the "water-accessible surface area," ASA. In problems involving folding energetics, ASA provides an indispensable link between the amount of buried nonpolar surface and the free energy change for burying that surface. Another major interest of Fred's in the '70s and '80s was the remarkably high packing densities of proteins. This interest dated back to his PhD research when he designed and built a special microbalance for measuring the density difference between a protein crystal and its mother liquor.13 In his computer analysis of packing densities,14 Fred found that proteins are packed as tightly as organic crystals. The average packing density of proteins is approximately 0.75, nearly the same as that of close-packed spheres.11 This finding was puzzling because the side chains of the nonpolar amino acids found in protein cores have irregular shapes: how could these side chains pack so tightly? In the mid-'80s Fred and Jay Ponder, a postdoc whose background lay in organic chemistry, found a way of phrasing this question that launched the study of the packing of protein cores. Their question was based on the inverse protein folding problem: how many amino acid sequences are compatible with a given backbone structure (or tertiary fold)? They asked how many variations of the core residues are possible if the core must be tightly packed, the backbone is fixed, and certain other restrictions apply.15 These constraints give a huge reduction in the number of possible core sequences and Ponder and Richards proposed that packing the core is a main factor determining the protein's tertiary fold. Their paper aroused tremendous interest in the protein folding community, which had become sizable by 1987. Several workers set out to test their proposal (see Ref. 16), and the consensus view emerged that the flexibility of packing protein cores is more forgiving than envisaged by Ponder and Richards. However, when Kuhlman and Baker17 re-opened the problem by using an algorithm based on folding energetics rather than geometry, they found that relatively few sequences are consistent with a fixed backbone conformation for any of the proteins they tested, and this is especially true for core residues. Their results agree with the Ponder and Richards proposal and the problem of packing protein cores is very much alive today. I've chosen only a few of Fred's many research interests for discussion. His memoir5 examines more of them, but by no means all. Frederic M. Richards died of natural causes on January 11, 2009. He had been in failing health for some time. He left a wife Sarah ("Sally"), a son George, and two daughters by a previous marriage, Sarah and Ruth. The family has set up a blog for recording anecdotes about Fred at fredericmrichards.blogspot.com. The author is grateful to the following people for information, anecdotes, and discussion about Fred: David Davies, Mark Hermodson, Brian Matthews, Jay Ponder, George Rose, John Schellman, Dieter Söll, Tom Steitz, and Lubert Stryer.