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Autobiography of J. Michael Schurr My parents were born and raised in the Pacific Northwest and were college graduates. They first met in the mid-1930s while working on the east coast. They eventually married in Massachusetts and completed their family with the arrival of me in 1937 and my sister in 1938. My dad worked for General Electric at the time, but soon joined Kaiser Industries for whom he worked at various locations on both coasts during WWII. For the first 11 years, my geographic trajectory resembled the Brownian motion of a particle in a weak potential well centered at Seattle, where we eventually settled. My interest in biology developed from an early age. I spent a lot of time with my grandmother, who lived in a small waterfront cottage on Bainbridge Island with a typically organic Puget Sound beach. In addition to many species of fish and clams, the profusion of marine animals on the beach and in the shallows included crabs, barnacles, starfish, sand worms, sand dollars, sea cucumbers, and both orange and clear jellyfish, the latter of which phosphoresced when disturbed during a late summer’s night. My acquaintance with such exotic creatures raised many questions. Most were directly biological, pertaining to how these organisms ate, breathed, moved, grew, and reproduced, and their answers were already known to biologists. Some questions were more chemical or physical in nature. For example, how do barnacles, mussels, and kelp attach themselves so firmly to otherwise slippery marine rocks? Why do fish secrete slime and what is it? How do jellyfish phosphoresce? These latter questions had no good answers at the time, and many years would pass before they did. Any new knowledge only raised more questions. I knew that salmon returned up tiny streams to their birthplace, but how did they recognize and remember their particular home stream? The signals governing salmon navigation were later shown to involve olfaction, but the chemical agents involved and the mechanism of memory imprinting of the smolts still remain unidentified. One could justifiably conclude from the foregoing that it is far easier for even a child to ask basic physical or chemical questions pertaining to living things than it is for highly trained scientists to answer them. As a youth, certain physical laws crept into my consciousness in an entirely empirical way. My early intuition regarding the persistence of linear momentum and its changes under applied forces was derived largely from throwing, hitting, and catching balls, stones, sticks, and other objects. My awareness of Newton’s third law was derived from numerous failed attempts to dive significant distances off of paddle boards or dinghies, which produced practically no net displacement from my starting position. My epiphany regarding the conservation of angular momentum came in third grade and was a consequence of failing to take it into account while attempting to replicate a Tarzan move I had seen at the cinema, but with a far shorter pendulum rope. Casts on both wrists and a sling to relieve the strain on a damaged shoulder allowed me plenty of time to contemplate the omission of angular momentum from my initial feasibility reckoning. My formal training in science began in high school where three extraordinary people introduced me to mathematics, biology, chemistry, and physics. In addition to the regular curriculum in grades 9 and 10, Martha Hardy propelled a few of us through analytic geometry and elementary differential and
integral calculus. In grade 10, Art Haines disclosed the deepest secrets of countless organisms small and large and then unveiled ecology, genetics, and Darwinian evolution. Bob Whitney taught chemistry and physics in grades 11 and 12, respectively. Via insightful demonstrations, often using instruments of his own construction, he enabled us to observe at close range many intriguing chemical and physical phenomena that we could never have seen otherwise. His classes planted the captivating idea that chemical and physical phenomena might actually be understandable in terms of simple rules, even though many of the rules were not yet apparent to me. In high school, I worked at odd jobs but also found time to participate on the high school ski and tennis teams. I attended Yale University on a scholarship with the intent to become a physician and scientist. My major was biophysics, which meant that I took lots of physics and biology courses, but only two in chemistry. I also worked in a radiocarbon dating laboratory for two years. My coursework sparked certain latent interests. I had been fascinated by Brownian motion, ever since I had first witnessed it in a microscope in high school, and was delighted to learn about Langevin’s theory. It seemed miraculous that one could apply Newton’s equation knowing only the drag forces, but without knowing in detail the potentials involved in the fluctuating solvent forces, and come up with the diffusion coefficient, as Langevin had done. I sensed that something important must be hidden from view, but couldn’t put my finger on it. I was also enthused by rotational Brownian motion, and wrote a paper summarizing Perrin’s early work on fluorescence depolarization for a biophysics class. By the end of my junior year I had become increasingly attracted to the study of fundamental science. After observing my professors, I realized that a career of teaching and research in any fundamental science would require such an immense effort that no time would be left for treating patients. During my senior year, I incorporated graduate physics and mathematics courses into my curriculum, and decided in favor of graduate school at UC Berkeley over an MD-PhD program at Stanford. After my first year at Yale, I developed a romantic interest in a wonderful young woman and fellow skier from my high school class, Karen Martin. It was largely a correspondence courtship since she was at Stanford when I was at Yale, and when she was at home I was working for the U.S. Forest Service south of Mt. Rainier. Karen and I married before the start of my senior year, and we have been together ever since. Incredible though it may seem, she still laughs at most of my feeble jokes. During the summer following my graduation from Yale, I joined an ecology group at Oak Ridge National Laboratory. I worked closely with Maynard Stamper, a field biologist and crayfish expert, to investigate the uptake of strontium and calcium by recently molted crayfish. This was part of a larger program to track the accumulation of radio-nuclides in the food chain. Our studies advanced far enough to propose a quantitative model for the observed asymptotic uptake, and we eventually published a paper, my first in a refereed journal. I entered the Graduate Biophysics Program at UC Berkeley. Biophysics was then a new and somewhat ill-defined area. Most biophysicists were either radiation biologists or electro-physiologists, and most did not work at the molecular level. I wanted to study the physical chemistry of biological macromolecules,
10.1021/jp810594a CCC: $40.75 2009 J. Michael Schurr Published on Web 02/26/2009
2546 J. Phys. Chem. B, Vol. 113, No. 9, 2009 especially those phenomena not exhibited by smaller species, such as thermal denaturation transitions, the formation of stable well-defined supramolecular aggregates, and properties associated with their often very large intrinsic charges. I also had a hunch that the deformational dynamics of biological macromolecules in solution might differ significantly from those of small molecules, possibly exhibiting time-dependent elastic moduli, something like silly putty. At the time, I didn’t know enough to read the extant literature in an informed way, so I spent my first two years taking mostly graduate courses in several departments. I first joined the group of Chet O’Konski in the Chemistry Department, who studied the ion-atmosphere dynamics of colloidal spheres and Tobacco Mosaic Virus. After several months of charged colloidal spheres, I switched advisors and joined the group of Doug McLaren in the Department of Soils and Plant Nutrition. He was interested in the activity of enzymes in more realistic environments than free solution. There I performed a variety of experiments to identify some of the parameters that affect the rates of digestive enzymes (trypsin, subtlisin, and pepsin) acting on structured substrates. Most informative was a comparison of the rates of tryptic hydrolysis of gelatin in free solution and in gel-microspheres over a range of temperatures below and above the melting point. During the long kinetic runs, I found time to investigate the role of diffusion in reVersible bimolecular reactions by developing a theory to treat both the forward and backward reactions simultaneously. This theory was extended to analyze enzymatic reactions and was published with some additional work done at the University of Washington (UW) in 1970. I have returned occasionally to diffusion-controlled reactions and still have some work to write up. At UC Berkeley, I became acquainted with several contemporaries, who later pursued successful careers as research scientists, and with whom I have subsequently interacted scientifically: Bob Glaeser and Paul Todd in biophysics; Nancy Stellwagen in chemistry; Tony Warren in biochemistry; and Ju¨rg Ruchti in soils and plant nutrition. Both in and after graduate school I learned valuable things from all of these folks. I arrived at the University of Oregon for postdoctoral study in the group of Bill Simpson in the Chemistry Department. Bill was a quantum chemist and spectroscopist who was interested in the coupled tautomeric resonance of the protons in formic acid dimer and eventually in DNA base-pairs. Although I invested a lot of time in that project, the necessary computer power was still several years away, and I eventually abandoned it. Bill had developed a nonrelativistic formulation of quantum radiation-field theory suitable for dipolar sources. I used that to develop a general theory for the temporal dynamics of various initially prepared quantum states of the joint molecule-radiationfield system. Properties of the molecules, such as their energy levels and transition dipoles, were simply assumed. I was intensely curious to see how a number of phenomena/problems could be understood, when a full account was taken of the quantum nature of the radiation field. These included the following: the retarded interaction between two molecules, one of which is excited; under what conditions classical coupled oscillator theories for the optical properties of chromophore aggregates are valid; radiationless transitions; the response of an isolated chromophore to resonance radiation; light absorption at high intensities; the response of a chromophore to a short multiphoton pulse of radiation as a function of the off-resonance; and the Beer-Lambert law, which was actually the most difficult to derive. These studies, which spanned a decade, gave me a valuable sense of when it was or was not important to employ the full quantum theory of the radiation field. I have
continued to interact with two members of the Simpson group, Pat Callis and Curt Johnson, for many years and have learned much from each of them. I had always wanted to return to the University of Washington (UW), but that seemed less likely than winning the state lottery. By a miraculous process involving bad and good luck in the right order, I was hired into the Chemistry Department at the UW as its first biophysical/macromolecular physical chemist. During my interview, Dave Ritter, a boron chemist, said “I don’t know what a biophysicist is, but what I want to know is whether you are a dead one or a live one.” He wanted to be sure that I would actually be working on molecules and not on rats. Technological advances put amazing new tools in the hands of biophysical chemists of my generation. Analog circuits were replaced by fast digital devices of all kinds, including logic chips, sample-and-hold circuits, and transient recorders, and the speeds and digital capabilities of oscilloscopes rose rapidly. The speeds and memories of digital computers also soared, and the development of numerical algorithms, such as fast Fourier transform routines, proceeded apace. The appearance of fast minicomputers for online data acquisition and processing in the late 1960s really changed everything. Entirely new kinds of experiments became possible, wherein one could acquire, store, and process measurements at rates up to a million times per second. Both continuous wave and pulsed lasers had been invented, and the first commercial models were reaching the marketplace. Another technological advance was solid-state peptide synthesis. Barbara Shaw wanted to study the triple-strand association of collagen model polypeptides of controlled composition and undertook solid-state syntheses of (pro-pro-gly)n, n ) 7 and 8. Both species formed triple-helices at low temperature. Their equilibrium melting curves together with literature curves for the n ) 10, 15, and 20 peptides were simultaneously “fitted” by different models, which were evaluated via a grand partition function binding theory, to extract the best-fit common set of thermodynamic parameters. The late Leon Slutsky, my faculty mentor in the early days, had an extraordinary intellect, and in no small way influenced my thinking about many aspects of science. Because we were both too busy at school, our most extensive discussions took place while out climbing or skiing together in the mountains. Leon used ultrasonic attenuation to study the kinetics of proton transfer reactions. With his encouragement, my earlier theory for diffusion-controlled reversible reactions was extended to extract the response to a small-amplitude oscillation of the intrinsic rate constants. The shape of the predicted ultrasonic relaxation differed, but only modestly, from the usual Debye type. Leon and I coadvised Bob White, who investigated the kinetics of proton transfer reactions in several systems including hemoglobin, which involved about 100 simultaneous reactions. Together Bob and I developed the relevant theory of acoustic absorption for an arbitrary number of simultaneous reactions. The results were applied to analyze their hemoglobin data and published by Leon and Bob in 1972. Dynamic light scattering (DLS) was invented by in the mid1960s by Norm Ford and George Benedek at MIT. They measured the power spectrum of the fluctuating photomultiplier current with a radio frequency spectrum analyzer from which they obtained the dynamics of concentration fluctuations near a critical point. Their use of fluctuations in the intensity of scattered light was a dazzling trick to increase the effective spectral resolution by many orders of magnitude. Benedek’s group also measured the diffusion coefficients of a few
J. Phys. Chem. B, Vol. 113, No. 9, 2009 2547 macromolecules by the same DLS technique. It occurred to me that DLS might also be used to study rates of reaction, as well as rates of rotational diffusion (via the depolarized component of the scattered light) and even rates of macromolecular deformation. However, the data collection times of the original experiments were rather long, because only a tiny fraction of the photocurrent passed through the filter of the spectrum analyzer at any given time. Several folks, including me, realized that the efficiency of data collection could be increased by ∼100fold or more by collecting the entire fluctuating photocurrent record directly in the time-domain and processing that to compute its autocorrelation function (ACF), which was the primary quantity of interest in any case. I wanted to perform DLS experiments in the time-domain by using a minicomputer to record and process the fluctuating photocurrent, but I had neither a laser nor a minicomputer and virtually no money. My colleague, Lou Crittenden, and I won an intramural UW grant to acquire a PDP-12, the first minicomputer in our department. I also worked with another colleague, Dave Eggers, to build a He-Cd laser. Although it lased, its intensity fluctuated so much that it dominated the fluctuations in scattered light. Warner Peticolas at the University of Oregon learned of my predicament and generously offered to loan me his surplus Spectra-Physics He-Ne laser for which I remain forever grateful. Our vibrationisolation table consisted of ∼12 feet of 36-in. wide-flange steel I-beam, weighing about 3000 lbs, mounted on springs of my design. Its total cost was $140. With a lot of help from Sheldon Danielson and others in our electronics shop, and numerous folks in the machine shop, Ken Schmitz and I finally were able to perform DLS experiments. We set out to investigate the depolarized component of the forward scattered light from Tobacco Mosaic Virus and DNA. This was a challenging experiment, requiring exceptional optical quality of the polarizers and the flat end-windows of the cylindrical scattering cell. Indeed, only by locating sweet spots in all the optics, could we perform the experiment in homodyne mode. Building vibrations were a major problem. Many times I thought our instrument had a better future in seismometry than in depolarized DLS. Our experiments on Tobacco Mosaic Virus gave rotational diffusion coefficients for the monomer in good agreement with previous electric birefringence and dichroism measurements from other laboratories. Unfortunately, no evidence of bending motions was detected. Some 20 years later, Jess Wilcoxon, Lu Song, Ug-Sung Kim, and I finally succeeded in measuring the bending rigidity of a much longer and thinner filamentous virus, M13, by polarized DLS. Our forward depolarized DLS studies of high molecular weight DNA yielded a great surprise. The temperature profiles of the longest decay time in the ACF and the intrinsic viscosity together implied unequivocally that DNA molecules associated to form cross-linked bundles of much higher molecular weight in the upper melting region before finally dispersing at higher temperature. In order to overcome the loss of entropy upon association, there had to be an increase in entropy somewhere, but where was it? Several years later, John Thomas and Guy Fletcher in Australia performed forward depolarized DLS studies on triple-strand collagen. Their data likewise implied the association of triple-strand collagens to form large aggregates in the upper melting region before dispersing. Eventually, John Shibata and I theoretically treated a DNA equilibrium involving single strands, duplexes, quadruplexes, and hexaplexes with appropriate internal pairing rules. Empirical values were employed for the relevant thermodynamic parameters. I did not really believe that this would go anywhere and was very
pleasantly surprised when it did. With increasing temperature, duplexes predominated below the melting region, but eventually quadruplexes and later hexaplexes predominated in the upper melting region, and finally single strands emerged at still higher temperature. Had we been able to include them, somewhat larger complexes would surely have predominated for the longer DNAs. For collagen, a corresponding treatment of single strands, triplexes, and hexaplexes produced similar behavior. The entropy driving these associations is just the tremendous number of configurations that result from the swapping of pairing partners within the multiplexes. Even though any typical hexaplex configuration is far less stable than any typical quadruplex configuration, there are far more possible hexaplex configurations, and the same holds for the comparison of quadruplexes and duplexes. This association phenomenon diminishes with decreasing chain length and is not significant for DNAs with less than about five melted regions, which is where the strand swaps are allowed to occur. This phenomenon seems to be general and should be exhibited by any multistrand biopolymer, or indeed by any polymer whose subunits have a saturable capacity for cross-linking to other strands, even when the crosslink is a ligand that must first be bound. Assembler code programming never seemed to end and was the bane of my existence for almost a decade. Numerous improvements to our instrumentation were made throughout the 1970s, many of which required me to write and debug new assembler code for the PDP-12. Besides having different number conventions (ones- versus twos-complement) in its two modes of operation, it had no hardwired “divide” instruction, so a subroutine had to be written for that, as well as for every mathematical function I used. The term “user hostile” comes to mind. During the decade of the 1970s, our intensity ACFs were obtained by four very different methods, before finally in the early 1980s the digital photon correlator of my dreams was designed and constructed by Jim Gladden and others in our electronics shop and the need for new assembler code abated. In the early to mid 1970s, Wylie Lee and Sung-Chan Lin undertook our first studies of poly-L-lysine (PLL). Their work revealed three separate phenomena, all stemming from the fact that PLL is a strong polyelectrolyte. (1) The apparent diffusion coefficient of the PLL increased 4- to 5-fold as the salt concentration was decreased from 1.0 M down into the 0.001-0.01 M range, due to interpolyion repulsions, and the scattered intensity similarly fell off. These results were consistent with the simple coupled mode theory proposed to analyze the data. (2) At salt concentrations below 10-3 M, there appeared only a very slowly relaxing diffusive mode associated with rather weak scattering, which we dubbed the extraordinary mode. This extraordinary mode was confirmed for PLL and other linear polyions in other laboratories and was later characterized in detail by Marian Sedlak in Kosice. However, a satisfactory theoretical description is still lacking. (3) The diffusion coefficient of isolated polyions declined with decreasing salt concentration to values well below what could be attributed to hydrodynamic drag alone. This was ascribed to electrolyte friction, and a simple theory was proposed to account for that. Substantial improvements in the theory were later effected by Magdaleno Medina-Noyola and co-workers at San Luis Potosi. Polarized DLS studies of the internal dynamics of DNA were pursued in different contexts for almost 20 years. The first step was to develop a theory for the dynamic structure factor of the Rouse-Zimm (RZ) bead-and-spring model. By proceeding directly from the coupled Langevin equations and Gaussian character of the fluctuating forces, the dynamic structure factor
2548 J. Phys. Chem. B, Vol. 113, No. 9, 2009 could be formulated and then simply squared to obtain the relaxing part of the intensity ACF. The second step was to develop a capability to perform DLS measurements using 251 nm radiation in order to extend the range of accessible scattering vectors and thereby probe motions on distance scales down to ∼22 nm. The third step was to find ways to clean up our samples and to assay for protein/polyamine contaminants. The ensuing studies showed that any local opening of the DNA at ∼20 °C at neutral pH is so rare as not to contribute significantly to its bending or torsional flexibility. Even occasional single strand breaks have no effect. However, at pH 10, single-strand breaks, contaminating proteins, added polyamines, and covalently bound putrescines all introduce titratable joints, wherein the DNA is opened and a local torsional rigidity weakness is created. This work was carried out over 6-7 years by Sung-Chang Lin, Stuart Allison, John Thomas, and Jess Wilcoxon. My colleague, Bruce Eichinger, provided invaluable theoretical assistance, and another colleague, Dave Teller, provided crucial sedimentation measurements. We turned to the time-resolved fluorescence polarization anisotropy (FPA) of intercalated ethidium as a means to measure directly the torsional rigidity of DNA. The first step was to develop a theory for the FPA of a chain of rigid disks, each carrying a transition dipole and connected to its neighbors by torsion and bending springs. Although the torsional contribution was easy, because of its similarity to the dynamic structure factor of the RZ model, a correct formulation of the FPA in the presence of significant bending took somewhat longer, and an accurate account of the bending contribution for weakly bending rods took longer still. The second step was to build an instrument for time-resolved FPA measurements around our department’s Spectra-Physics synch-pump dye-laser system. At the time, it was an amazing light source, but it consumed expensive plasma tubes like a kid eating popcorn. This pulsed laser facility was obtained in 1979 via a successful NSF instrumentation proposal, and its main users were Bill Parson’s and my groups. Together we operated, upgraded, and shared that facility for 15 years. FPA measurements on many different DNAs indicated a more or less uniform torsional rigidity that depended only weakly upon overall percent GC composition, but varied somewhat more significantly with sequence. The torsional rigidity proved to be a sensitive probe for changes in secondary structure, such as the introduction of titratable joints. A remarkable new phenomenon emerged in our first studies of a supercoiled DNA. Its secondary structure switched in response to changing the buffer from Tris to citrate, and the associated decreases in torsional rigidity and circular dichroism (CD) spectrum were surprisingly large. In contrast, linear DNAs were insensitive to such buffer changes. Then, varying the superhelix density of another supercoiled DNA from 0 to native (-0.05) gave evidence for two structural transitions. Even more remarkable, a local perturbation, namely binding a transcriptional activator protein to its specific site on the DNA, caused a change in secondary structure that apparently extended over a large (g400 bp) tract of its flanking DNA. This was observed for two different transcriptional activators, one bound to a supercoiled DNA and the other to a linear DNA. Equally remarkable, inserting a 16 bp (CG)8 sequence near the middle of an ∼1100 bp linear DNA altered a large (g400 bp) tract of its flanking DNA. When the insert switched from right-handed to lefthanded with increasing salt concentration near 2.5 M salt, the structure of its flanking DNA also changed (again). When 1 ethidium/300 bp was added to the solution, it changed a third time. The conclusion that secondary structures of at least some
DNA sequences are not only switchable, but switchable over rather long distances from the site of a local perturbation, now seems inescapable. In 1984, we proposed that a regulatory protein, upon binding to its specific site, could alter the secondary structure at a distant RNA polymerase site via a long-range, allosteric transition in the structure of its flanking DNA. Requirements for such a phenomenon are that the DNA must exhibit two or more distinct secondary structures, that their free-energies per base-pair must be rather similar, and that the transition between them must be sufficiently cooperative to attain a large domain size. The results described above are consistent with this proposal, which unfortunately also has a down side. The kinetics of structural change in any highly cooperative system is expected to be rather slow in the absence of a coercive force, so that metastable states and ultraslow equilibration rates might be encountered. In fact, our group has repeatedly encountered such behavior. Immediately after the release of superhelical stress in supercoiled DNAs, the secondary structure initially resides in a state with an anomalously low torsional rigidity for up to a week or more, before it begins to rise up toward the equilibrium value, which may take several more weeks. This is true, whether the superhelical stress is relaxed by cutting both strands, by intercalating sufficient ethidium or chloroquine (which unwind the DNA), by binding single-strand binding proteins (which partially melt the DNA), or by Topoisomerase I (which relieves superhelical stress and reseals the circular DNA). The last experiments before my laboratory closed indicated that excess Topoisomerase I (Topo I) can equilibrate the initially formed metastable secondary structure! Things would have been much easier had we known this 25 years ago. The above work spanned about 30 years, and was carried out by Stuart Allison, John Shibata, John Thomas, Jess Wilcoxon, Steve Benight, Jo¨rg Langowski, Bryant Fujimoto, Lu Song, Peng-Guang Wu, Jim Clendenning, Ug-Sung Kim, Jeff Delrow, and Greg Brewood with help from Richard Humbert and my UW faculty colleagues, Clem Furlong and Dave Teller. By 1988 several observations suggested that the bending rigidity manifested in optical anisotropy measurements on DNA was much greater than its equilibrium value, which corresponds to a persistence length, P ) 50 nm. We considered the possibility that the dynamic bending rigidity on a short time-scale, less than 10 µs, might substantially exceed the equilibrium value. Early estimates suggested values of the corresponding dynamic persistence length in the range, Pd ) 125 to 210 nm, and we began using Pd ) 150 nm to extract torsional rigidities from our FPA measurements. Several years later, we undertook a transient polarization grating (TPG) experiment to measure the optical anisotropy of a 200 bp linear DNA from 0 to 10 µs. The combined TPG and FPA data on the same DNA enabled us to determine its dynamic persistence length, Pd ) 200 nm, its equilibrium persistence length, P ) 50 nm, and its torsional rigidity, C ) 188 fJ fm. Comparably large values in the range Pd ) 150-170 nm had just been obtained via EPR spin-label methods by my colleagues Bruce Robinson and Paul Hopkins and their students. Evidently, the bending rigidity of DNA is a relaxing quantity that declines over time to approximately onefourth to one-third of its initial value. This implies that a slow equilibrium between two or more states with different intrinsic curvatures prevails in DNA but is effectively frozen on the 10 µs time scale. Slow bending allows population to shift from one state to the other, which reduces the effective bending rigidity. A corollary of this finding is that sufficient imposed
J. Phys. Chem. B, Vol. 113, No. 9, 2009 2549 bending strain should shift the equilibrium between different secondary structures. In fact, when a 181 bp DNA is circularized, its torsional rigidity and intrinsic binding constant for ethidium both jump to higher values, indicating a change in average secondary structure due to the imposed curvature. Evidence for relatively slow structural fluctuations at TpA steps of small duplex DNAs came from NMR studies of Mike Kennedy and Kate McAteer at the Pacific Northwest National Laboratory. They found anomalously large linewidths of a particular adenine proton at all TpA steps. Our own NMR relaxation measurements on a site-specific 13C-labeled thymine at a single TpA step suggest that more than two states are involved and establish that the time-scale for interconversion exceeds 0.03 s. The torsional rigidities obtained from FPA measurements are insensitive to the choice of Pd in the range 150-200 nm, and for unstrained or only weakly strained DNAs lie in the range, C ) 170-210 fJ fm. An alternative way to estimate the torsional rigidity is first to measure the twist energy parameter, which is proportional to the Hooke’s law torque constant for supercoiling, via biochemical methods, and then to perform Monte Carlo simulations and reversible work calculations of the twist energy parameter for various trial values of the torsional rigidity. Very long simulations, which are now feasible, have reduced statistical errors in the computed twist energy parameters and enabled us to narrow considerably the range of possible torsional rigidities that could give rise to the measured values. The allowed range admits the values mentioned above, but specifically excludes the substantially higher values measured for small circular DNAs or DNAs under considerable tension in single-molecule experiments. Those evidently do not apply to unstrained or only weakly strained DNAs, and presumably stem from structural responses to the imposed bending strain or tensile stress.
We recently identified a full sigmoidal transition between two duplex states with different torsional and bending rigidities within the B-family, which is induced by ethylene glycol. The cooperativity almost certainly exceeds 30 bp and could be far larger. The above work spanned about 20 years and was performed by Lu Song, Alexei Naimushin, Pat Heath, Roy Diaz, John Gebe, Hazen Babcock, Jeff Delrow, David Rangel, Chris Sucato, Greg Brewood, and Bryant Fujimoto with theoretical assistance from Stuart Allison. Alas, I have arrived at the end of my academic career, while still only at the beginning of what promises to be a long and fascinating story pertaining to induced structural transitions in DNA and their possible roles in biology. The most rewarding part of my job was interacting with my junior collaborators. I sincerely thank them all for their many contributions to our joint enterprise. I am also grateful to my faculty colleagues past and present, who have provided a wonderfully supportive and stimulating environment in which to work. I particularly thank Bruce Eichinger for my education in polymer physical chemistry, and Bruce Robinson and Gary Drobny for my education in magnetic resonance. I am especially grateful to Bryant Fujimoto, my close collaborator for over 20 years, for his knowledge and skill in so many areas and his ability to keep forging ahead. Finally, I thank Steve Benight for arranging this special issue. In my other life, Karen and I raised two lively daughters, and eventually acquired two equally lively sons-in-law, four very lively grandkids, and a mountain of treasured memories.
Mickey Schurr JP810594A
