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Tribute to William A. Eaton
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MOLECULAR SPECTROSCOPY OF HEMES In the 1960s, the subtleties of quantum mechanics and spectroscopy were lost on most biophysicists. Bill was different from most. He is a master of spectroscopy. Using a combination of methods more familiar to solid state physicists, such as polarized single crystal absorption and natural and magnetic circular dichroism, along with the emerging tools of molecular orbital and crystal field theory, Bill laid the foundation for understanding the complex electronic absorption spectra of heme-containing proteins. His early spectroscopic studies laid the groundwork for understanding the photophysics of ligand dissociation, beginning with one of the first applications of picosecond time-resolved spectroscopy in biology in collaboration with Robin Hochstrasser. These processes were among the first to confront the field of molecular dynamics simulations of biomolecules, which were limited to short time scales and thus often safe from test.
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MYOGLOBIN, HEMOGLOBIN, AND ALLOSTERY A lot of picoseconds elapse before most biologically interesting molecular events take place. With Eric Henry, Jim Hofrichter, and postdoctoral fellows Anjum Ansari and Stephen Hagen, Bill began to fill in the gap by studying myoglobin dynamics using nanosecond-resolved optical spectra. The experiments showed the complex dynamics seen at low temperature were also present in a room-temperature trehalose glass. The results demonstrated that internal friction was a dominant contribution to conformational relaxation following photodissociation of the carbon monoxide (CO) complex. Internal friction has since become a recurring topic in protein folding and dynamics. Bill’s work on hemoglobin was even more influential. While the oxygen/hemoglobin system is the poster child for allostery in multisubunit proteins, the field was rife with speculation until Bill’s experimental work. From elegant oxygen binding measurements on single crystals of hemoglobin using polarized light with Andrea Mozzarelli at the University of Parma, Bill settled the 25-year-old controversy of whether oxygen binding required a change in quaternary structure, as in the allosteric model of Monod, Wyman, and Changeux (MWC). Bill then explained how the deficiencies of the MWC model in accounting for the regulation of oxygen affinity by the binding of small molecules at sites distant from the hemes (so-called allosteric effectors) could be overcome using a model he developed with Henry and Hofrichter in which there is a functionally important preequilibrium between two tertiary structures, as well as the two quaternary structures proposed in the MWC model.
Ernie Branson, Photographer
diting this special issue of The Journal of Physical Chemistry B in honor of our friend and colleague, William Eaton, on the occasion of his 80th birthday, is a great pleasure and privilege. Bill’s pioneering work and his intellectual driving force have had a profound impact on the development of protein physical chemistry and biological physics. His early spectroscopic investigations laid the foundation for studies of hemeprotein dynamics; his studies of sickle cell hemoglobin polymerization resulted in a totally new approach to understanding the pathophysiology and therapy of sickle cell anemia, the paradigm molecular disease; his studies of the equilibria and dynamics of hemoglobin took our understanding of allostery to a new level and settled a 25-year controversy on the mechanism of oxygen binding; he initiated the field of ultrafast protein folding by improving the time resolution of folding studies by 5 orders of magnitude with laser triggering; and together with his pioneering use of single-molecule fluorescence methods, he opened completely new experimental avenues to fundamental processes in protein folding dynamics. In all of this work, he closely combined experiments with theoretical modeling and simulations, bringing together the experimental and theoretical communities to understand how proteins work. With his ability to identify the most important issues and his relentless pursuit of a truly quantitative understanding of complex biophysical problems, Bill has set the agenda for others to follow, time and time again. In what follows, we outline only a few of the many facets of Bill’s research.
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© 2018 American Chemical Society
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SICKLE CELL HEMOGLOBIN FIBER FORMATION Sickle cell anemia was the first disease to be traced to an individual molecule. However, the underlying physics was unclear, to say the least. Bill entered the sickle cell field by obtaining Special Issue: William A. Eaton Festschrift Published: December 13, 2018 10971
DOI: 10.1021/acs.jpcb.8b08745 J. Phys. Chem. B 2018, 122, 10971−10973
The Journal of Physical Chemistry B
Special Issue Preface
important information on the structure of the sickle fiber using polarized absorption measurements on individual sickled cells. He then turned his attention to understanding the thermodynamics, kinetics, and mechanism of fiber formation. With Philip Ross and Jim Hofrichter, then a postdoctoral fellow, Bill discovered the highly unusual aggregation kinetics of sickle hemoglobin in slow temperature-jump experiments. They observed a long delay period prior to the explosive appearance of birefringence from fiber formation, with a delay time that depended inversely on the 30th power of the sickle hemoglobin concentration, the highest concentration dependence ever observed for a molecular process. To explain both the long delay period and its high concentration dependence, Bill, Hofrichter, and then postdoctoral fellow, Frank Ferrone, devised a new kind of mechanism that includes secondary nucleation on the surface of pre-existing fibers, which they called heterogeneous nucleation. One of the striking predictions of the double nucleation mechanism is that the delay time in small solution volumes fluctuates due to the stochastic appearance of the first single homogeneously nucleated fiber, which then goes on to form a large number of heterogeneously nucleated fibers that ultimately are detected by light scattering. These kinetic experiments were thus revealing single-molecule events, analogous to those in patch clamp experiments reported just a year or so before, in which opening of a single channel is not directly observed but is detected indirectly by the resulting macroscopic ion current. The model further predicts that, under certain solution conditions, the concentration dependence of the rate of homogeneously nucleating fibers should have an exponent twice that of the concentration dependence of the inverse delay time. Bill likes to point out that the 80th power dependence confirmed in this work is one of the most remarkable experimental results in all of chemical kinetics but nevertheless got the “grand slam”: Their paper, eventually published in the Biophysical Journal, was rejected by the editors of Nature, Science, PNAS, and Cell, without even getting sent out for review. Bill’s work represents the most thorough physical characterization of any protein selfassembly system to date, and the double nucleation mechanism is now widely used to explain the aggregation kinetics of the Alzheimer’s peptide and other amyloids. An extensive series of Bill’s studies together with Jim Hofrichter led to a rigorous thermodynamic description of a hemoglobin S gel at equilibrium and to a molecular model for the control of polymerization by oxygen. They perfected an assay, subsequently widely used in other laboratories, which determines the stability of the fibers from solubilities measured as the concentration of free hemoglobin molecules in the supernatant after sedimenting the fibers in an ultracentrifuge. Their experimental results on the solubility as a function of the saturation of the free hemoglobin molecules with oxygen could be almost perfectly explained by the simple postulate that only the T quaternary structure can enter the fiberall R conformations are completely excluded. Quite recently, Bill, Frank Ferrone, and Troy Cellmer made the remarkable discovery that, while there is no correlation of the delay time with solubility (which might be expected because of linear free energy relations between rates and equilibria), all of the data collapse onto a single universal curve when the supersaturation is defined as the ratio of the initial activities to the activity at equilibrium. The experimental work of Bill and Hofrichter in the 1970s also demonstrated an enormous contribution of excluded volume effects at the high protein concentrations required for
polymerization, still one of the most impressive examples of molecular crowding in cells. Because of his medical training, Bill immediately recognized the significance of the extraordinary kinetics of fiber formation for sickle cell disease. The delay time allows the vast majority of cells to escape the small vessels of the tissues (the microcirculation) before hemoglobin fibers form, making the disease survivable after adult hemoglobin has replaced fetal hemoglobin: The enormous kinetic sensitivity of the system accounts for the unpredictability and episodic nature of pain crises; increasing the delay time to allow more cells to escape the microcirculation prior to fiber formation would thus be therapeutic; and a large body of diverse clinical data could be understood and the severity of the disease determined by considering a single metricthe delay time relative to the transit time through the narrow vessels of the tissues. Factors which shorten the delay time, such as fever, or that increase the transit time, such as increased adhesion to the capillary endothelium by elevation in the number of white cells associated with infection, make the disease worse, while those that increase the delay time or shorten the transit time are beneficial. Bill’s experiments showed that, while almost every cell would be sickled at equilibrium and therefore occlude all vessels, the disease is survivable precisely because the delay period prevents most cells from sickling in vivo. He further proposed that small reductions in the concentration of sickle hemoglobin, by allowing more cells to escape the microcirculation before fibers form, would be therapeutic. Hydroxyurea, the only specific antisickling drug currently approved for treatment of sickle cell disease, in fact works according to the Eaton mechanism: It reduces the sickle hemoglobin concentration by inducing the synthesis of noncopolymerizing fetal hemoglobin. Bill has recently returned to his passion for sickle hemoglobin and is currently taking advantage of the clinical infrastructure at NIH to search for new drugs that increase the delay time by using a highly sensitive and automated laser photolysis-image analysis method that accurately measures the time that fibers take to form inside sickle cells. Using this assay, he and his co-workers have already discovered powerful inhibitors of fiber formation that act by producing small increases in cell volume to decrease the intracellular hemoglobin concentration, showing that increasing cell volume is a viable approach to therapy.
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PROTEIN FOLDING Among the younger generation, Bill is best known for his work on protein folding, and for changing experimental research in that field from biochemistry to biological physics. Prior to his introduction of optical triggering methods, a major experimental limitation in investigating the kinetics of protein folding was the poor time resolution of conventional methods, the most commonly used one being the stopped-flow technique, with a dead time in the millisecond range. This situation changed dramatically with the pioneering work of Bill, who introduced nanosecond pulsed laser techniques to trigger folding. Following this work, the development of a series of methods, including laser temperature jump, photoselection, and ultrarapid mixing techniques, gave the first glimpse of previously inaccessible elementary events of protein folding, such as the dynamics of the formation of secondary structure, loops, and the global collapse of the polypeptide chain. Bill’s studies using all of these tools now permitted not only the investigation of the mechanism of formation of the basic structural elements of proteins, but they also enabled the study 10972
DOI: 10.1021/acs.jpcb.8b08745 J. Phys. Chem. B 2018, 122, 10971−10973
The Journal of Physical Chemistry B
Special Issue Preface
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EATON, THE NIH CITIZEN Beyond his own research, Bill has been a strong advocate of expanding experimental, theoretical, and computational biophysical science at the NIH and has been a key player in recruiting scientists in these disciplines to several NIH institutes. He has also enriched the intellectual life in biophysical science at NIH by hosting distinguished scientists for extended visits, including H. Franklin Bunn, Alan Fersht, Quentin Gibson, Michael Levitt, and Peter Wolynes. As Chief of the Laboratory of Chemical Physics (LCP) in the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) since 1986, Bill was responsible for building the laboratory into one of the very top biophysical research groups worldwide. With the exception of Robert Zwanzig, who was already a legend in theoretical chemistry when he joined LCP, each investigator hired by Eaton went on to become a leader in his or her fieldAd Bax, Marius Clore, Angela Gronenborn, and Robert Tycko in nuclear magnetic resonance spectroscopy, Philip Anfinrud in timeresolved X-ray crystallography, and Gerhard Hummer and Attila Szabo in theoretical biophysics. His most recent hires, Robert Best and Hoi Sung Chung, are on a trajectory to becoming leaders in computational biophysics and single-molecule fluorescence spectroscopy. Bill’s impact has further been amplified by many of his postdoctoral fellows that are now on the faculty of research institutions in the US and Europe. Since 1986, Bill has been the Scientific Director of the Intramural AIDS Targeted Antiviral Program (IATAP) in the Office of the Director of NIH. This program, which is the first of its kind at NIH, received a special budget from Congress early in the AIDS epidemic to attract the best scientists at NIH to turn their efforts to basic research on the structural, molecular, and cell biological aspects of the human immunodeficiency virus in order to identify molecular targets for therapeutic intervention. Bill did a masterful job of getting this program quickly off the ground, putting his own research aside for many months. During the past 32 years, Bill has recruited outstanding investigators in structural biology to NIH to work on AIDS, has overseen the review of proposals and distribution of 2-year grants to NIH scientists from a wide range of disciplines, and has promoted many collaborations among NIH scientists through his annual IATAP workshops. Bill’s IATAP program is in part responsible for the sterling record of NIH scientists in meeting the AIDS crisis and is now being used as a model for new special granting programs within NIH in areas such as bioterrorism and orphan diseases. Bill’s impact on science has thus been profound, as a pioneer and trendsetter in diverse areas of biophysics, as an inspiring mentor and colleague, and as a visionary and architect of the Laboratory of Chemical Physics and science at NIH as a whole. His work already serves as an example to generations of researchers, experimentalists, and theorists alike, and his remarkable combination of innovation and rigor has made Bill an authority not only in his own field but far beyond. The impressive collection of articles in this special issue bears testament to the wide range of research done by his friends, colleagues, students, admirers, and competitors, all of which is linked to his interests and influence. Happy Birthday, Bill, and we hope you will enjoy reading your special issue!
of the full folding of the single-domain proteins that have been the object of intense investigation by molecular dynamics simulations. The beta hairpin study with postdoctoral fellow Victor Muñoz is particularly noteworthy, not only because it was the first equilibrium and kinetic study of an isolated beta hairpin but also because the recognition of the conceptual similarity between the physical chemistry of hairpin and single-domain protein folding made the experimental results on the hairpin one of the most popular benchmarks for testing molecular simulations. These studies of forming the basic structural elements of proteins led Bill to introduce the notion of a “speed limit” to protein folding. This concept caught on very quickly and has motivated the further search for ultrafast folding proteins and studies to re-engineer them to fold even faster by many research groups. Bill’s temperature-jump measurements on a double mutant of the villin subdomain still holds the folding speed record of 700 ns. His experiments on this protein, as well as earlier work on an α helix and the β hairpin, also introduced the concept of internal friction for the kinetics of protein folding. One of the most controversial aspects of protein folding mechanisms revolves around the uniqueness of a path to folding. This question is not accessible directly to bulk experiments. Starting in 2002, Bill has used single-molecule fluorescence methods to confront this issue. Bill’s group produced farreaching and influential results. A few of the “firsts” originating from his use of single-molecule fluorescence experiments include (i) showing with postdoctoral fellows Ben Schuler and Everett Lipman that the size of the chemically denatured state of proteins increases with increasing denaturant concentration; (ii) using microfluidics to study single-molecule kinetics under nonequilibrium conditions; and (iii) demonstrating with postdoctoral fellow Robert Best how to obtain accurate distance information from single-molecule FRET measurements by accounting for orientational and translational motion of the donor and acceptor fluorophores. Even greater things were to come when Bill focused on measuring the time it takes for a successful crossing of the freeenergy barrier separating the unfolded and folded statesthe transition path time. He recognized that all of the mechanistic information on how a protein folds and unfolds is contained in these rare and, experimentally, almost instantaneous events of a single-molecule trajectory. With his postdoctoral fellow, Hoi Sung Chung, now a principal investigator in the Laboratory of Chemical Physics, they employed a photon-by-photon analysis to determine the average transition path time of 1 μs for a small single domain protein. More interestingly, they found that the average transition path time is about the same for a larger protein that folded 10,000 times slower, a result predicted by energy landscape theory. The impact of this work has been far-reaching. Experimentalists using single-molecule force experiments seized on these ideas and quickly followed up on these works. Together, these studies have provided a completely new way of probing the folding of both proteins and RNA. In all of his folding studies, Bill paid close attention to theoretical developments by others, and developed statistical mechanical models to quantitatively describe their experimental data. Victor Muñoz and Bill developed a simple Ising-like model for protein folding that is capable of quantitatively explaining a wide range of equilibrium and kinetics results. This model, extended by Eric Henry, may be regarded as the simplest analytical model of protein foldingthe Hückel theory of protein folding.
Benjamin Schuler Attila Szabo Peter G. Wolynes
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DOI: 10.1021/acs.jpcb.8b08745 J. Phys. Chem. B 2018, 122, 10971−10973
