For Bil Clemons, the first glimpse of a protein structure never gets old. “Seeing the electron density for the first time—it’s still just magical and one of the greatest experiences you can have as a scientist,” says Clemons, a biochemist at the California Institute of Technology (Caltech).
Above: The crystal structure of Get4 bound to the N-domain of Get5 with the NMR spectra of the Get5 dimerization domain in the background. Image credit: Courtesy of B.Clemons
Those moments might be all the sweeter because of the months and years of trial and error that culminates in the final atomic image of a protein. As a graduate student, for example, Clemons put in 4 years to help solve the structure of the small ribosomal subunit, part of the molecular machine that translates genetic material into proteins in the cells. Now he works on labor-intensive membrane proteins.
Clemons got hooked on exploring the atomic terrain of molecules in part by a pair of 3D glasses when he was an undergraduate at Virginia Tech. The paper glasses were tucked into a special magazine on structural biology handed out in a class designed to help steer biochemistry students toward potential careers. Clemons ended up at the University of Utah in Salt Lake City. He was the first graduate student to join the lab of newly arrived Venki Ramakrishnan. Why did he make that choice? “I loved the concept of protein translation,” says Clemons.
About four years into Clemons’ PhD, Ramakrishan moved the lab to the MRC Laboratory of Molecular Biology (LMB) in Cambridge, England, largely because of the stable funding for risky projects like the ribosome structure. Clemons made the transition and helped to finish the atomic resolution structure of the 30S subunit. In 2009, Ramakrishnan shared the Nobel Prize in Chemistry for the structure of the ribosome. Notably, Clemons features prominently in Ramakrishnan’s 2018 memoir of the path to the Nobel, “The Gene Machine.”
As a graduate student, Clemons weathered the tedious task of freezing hundreds of crystals one at a time in the cold room by playing music. Clemons, who always has music on in the background, went through a Johnny Cash phase as a graduate student. But truth be told, the saxophone is the family instrument.
Clemons’ father and namesake, William, was a U.S. Marine bandsman and sax player who grew up in a segregated south. His late uncle Clarence was a saxophonist with Bruce Springsteen’s E-Street Band, a gig subsequently inherited by Clemons’ younger brother Jake. Clemons’ mother was an elementary school teacher from North Dakota. Because of the military, the family moved around, but one constant influence on Bil was a passion for understanding the natural world exemplified by the availability of iconic shows such as ‘Mutual of Omaha’s Wild Kingdom’.
When he received his PhD, membrane proteins were becoming a feasible area of structural biology. Contemplating a postdoctoral fellowship, Clemons emailed Tom Rapoport at Harvard Medical School, a leader in membrane protein biogenesis, for advice on what group would be best for working on the structure of the protein translocation channel. Rapoport asked for his CV, then quickly invited Clemons to join his team working on the SecY channel, a team that included Stephen Harrison and members of his group.
At HMS, Clemons and his colleagues determined the structure of the universal channel that allows newly made proteins to be transported through or into cellular membranes. The finding was reported in Natureon New Year’s Day 2004.
“The ribosome was great, because no one had conquered it,” Clemons said. “But we knew what that mountain looked like. The structure didn’t change the general basics of what we knew. But with the protein translocation channel, the structure told us everything. Leveraging the known biochemistry with a structure, we could finally generate mechanistic models that could be clearly tested. It has stood the test of time.”
Membranes create protected spaces for living cells with a bilayer defining an inside and outside. Embedded membrane proteins span this barrier and provide a critical path for information and nutrients. A third of the genome encodes membrane proteins. Most membrane proteins are made next to the membrane, because their hydrophobic sections are incompatible with the watery environment inside the cell. To ensure correct insertion, for most membrane proteins, the ribosome cozies up to the endoplasmic reticulum and pushes the protein through the protein translocation channel, where the protein can be inserted into the membrane. There, it can fold and then, if necessary, be trafficked to various organelles.
At Caltech, Clemons has been studying another pathway used by a class of membrane proteins, known as tail-anchored proteins, to integrate into the membrane. “They have a special toolkit for getting into the lipid bilayer,” he says.
The lab has structurally characterized the various components that take the protein from the ribosome and deliver to the endoplasmic reticulum through the cytoplasm. “It’s a dance, a series of handoffs of the proteins, orchestrated player to player,” he says.
In the process, he has run the gamut of structural biology methods and pretty much all the software packages at SBGrid. But structure is just the starting point for figuring out the mechanism for the proteins in motion. His team probes the dynamics and chemistry using biochemical and biophysical tools “to understand at the deepest level how they’re working,” he says.
In addition to his work on membrane protein biogenesis, the lab has focused on other areas, such as the twin-arginine secretion pathway and membrane associated glycosylation pathways.
The Clemons lab has added another tool: Computational biology. They are trying to take some of the trial and error out of the difficult early stage of studying membrane proteins. “Caltech has enabled me to lean on students with a lot of computational training,” he says. “With that, we’ve tried to think about problems in our area that haven’t been addressed and try to solve them.”
A big problem for structural biologists begins at the first step. Scientists often insert the gene for protein of interest into certain bacteria or yeast, and then induce the host to generate the protein. For membrane proteins, this is a make-or-break first step, because “it is unlikely that one can get enough protein to study,” Clemons says. “Every other step in the process has a high failure rate, but if you don’t succeed in the first step, you will not succeed at all.”
He proposed using computers to design a statistical model that would predict how bacteria will react when asked to create a protein they don’t normally produce. With a key graduate student, his team showed that it is possible to predict membrane protein expression directly from sequence data, which allows researchers to double their odds of expressing a membrane protein. The findings were published in March 2018 in the Journal of Biological Chemistry.
“It’s early days,” Clemons says. “But just to demonstrate that we can leverage computation for this complicated problem is exciting for us. Large data sets, statistical tools and bioinformatics are opening up new paths. The synergy between computation and biochemistry is very powerful.”
Until recently, a vaccinated llama has been a membrane protein crystallographer’s best friend. That was before Andrew Kruse and his co-authors showed that yeast can be a faster, cheaper, and possibly better tool for otherwise impossible crystallographic studies.
Broadly, the Kruse lab at Harvard Medical School is interested in how cells transfer information across their membranes. To probe the finer points of the molecular interactions, Kruse and his collaborators have developed new tools, including the yeast platform and a new evolutionary approach, to make studies of these membrane proteins easier for themselves and others.
Above: This structure of RodA in bacteria was solved using a de novo model constructed using sequence evolution analysis and may pave the way for next-generation antibiotics. Courtesy: A. Kruse.
About 20 years ago, it was discovered that the unique immune systems of camelids (llamas, camels, and alpacas) produce a pared down version of antibodies, featuring a single binding domain called a nanobody. Nanobodies were a boon to structural biologists. Among their many uses, these small molecules stabilize membrane proteins in specific different active conformations for crystallization and detailed structural analysis.
Yet despite the transformative impact on structural studies, it takes months to produce specifically tailored nanobodies by llama immunization. It can be prohibitively expensive, and the particles might not work. As a junior faculty member, Kruse felt constrained by cost and time limitations of immunization. So did a fellow junior colleague, Aashish Manglik at University of California, San Francisco. Together, the labs came up with a solution.
The yeast project arose out of their need for a better way to stabilize G protein-coupled receptors. GPCRs are a proteins lodged in fatty cell membranes that are crucial to human health and disease. The receptors detect hormones and neurotransmitters and then signal other proteins in the cell to respond. Millions of people take drugs targeting GPCRs, such as antihistamines, bronchodilators, antihypertensive agents, and many others. Better understanding of the structure and interactions could help researchers design more effective drugs with fewer side effects.
The yeast platform is one step toward the next stage Kruse envisions for GPCRs: A rational approach to finding and designing drugs to trigger or block specific activity.
“In signal transduction, there’s never been a more exciting time,” he says. GPCRs have both greasy and water-soluble parts, making them difficult to study at the atomic level until recently. The first high-resolution structure of a human GPCR wasn’t solved until the year 2007, Kruse says, but now dozens have been probed in their many different active shapes.
To boost those efforts, Kruse and his collaborators have made the vials of specially engineered yeast and instructions freely available to others for non-profit research. They fielded more than 100 requests from researchers in 17 countries little more than a month after publishing February 12, 2018, online in Nature Structural & Molecular Biology. About half of those requests came from posting the paper on the preprint service, BioRxiv. Other labs are using the nanobodies for projects ranging from identifying inhibitors of the zika virus to detecting bacterial species to classical genetic screens.
For as long as Kruse can remember, he dreamed of becoming a scientist. He grew up in the Twin Cities, Minnesota. Kruse discovered structural biology as an undergraduate at University of Minnesota, as a double major in biochemistry and math. It was a satisfying blend of fundamental quantitative biology with direct applications to human health. He worked in the lab of Douglas Ohlendorf on the structures of bacterial toxins.
Working in the lab also helped open up another part of the world. Because of its growing importance in science, Kruse decided to learn a Chinese language. A lab administrator connected Kruse to a university in northeast China where she had worked for years before settling in Minnesota. Before going to graduate school at Stanford, Kruse spent a summer working at the school in China. There, he met his wife. They speak mandarin exclusively at home. He has no formal collaborations in China, but an acquaintance at the National Center for Protein Science in Shanghai is helping distribute his new nanobody yeast platform within the country.
Kruse began working on GPCRs in graduate school in the Stanford University laboratory of Brian Kobilka. About halfway through Kruse’s PhD training, Kobilka shared the 2012 Nobel Prize in Chemistry with Robert Lefkowitz of Duke University for their discoveries on GPCRs.
In Kobilka’s lab, Kruse worked on a GPCR group called muscarinic acetylcholine receptors. In studies to understand the molecular basis for therapeutic drug action, he showed how tiotropium bromide (Spiriva), an inhaled drug for chronic obstructive pulmonary disease and for uncontrolled asthma, recognizes and blocks the muscarinic 3 (M3) receptor. The once-daily dose is effective in dilating bronchial airways for 24 hours, because the receptor closes around and locks onto the drug, trapping it in place, he found.
He also worked on the related M2 muscarinic receptor, showing how multiple compounds could bind simultaneously to the same receptor. In other projects, he and his Stanford co-authors developed a way to generate more selective affinity reagents for transmembrane proteins. The new method led to a patent and then to a company. In 2016, Kruse was selected by Forbes magazine for its 30-under-30 in health care, along with Aaron Ring, now at Yale University, who together had founded Ab Initio Biotherapeutics, Inc. Ring also collaborated with Kruse and Manglik, another co-founder, on the new yeast platform.
At Harvard, the Kruse lab continues to work on membrane signaling with a variety of approaches, both old and new. For example, they collaborated with another Harvard lab on a mass spectrometry technique to track signaling in living cells.
For a 2017 PNAS paper, they reached back a century to the days when medical researchers used material from nearby slaughterhouses. With a modern twist, they used mail-order frozen calf livers to track down the identity of Sigma 2, a receptor implicated in Alzheimer’s disease, schizophrenia and cancer. Despite its potential therapeutic importance, it had not been cloned. In this case, the lab purified the receptor from homogenized calf livers and revealed its identity as the transmembrane protein (TMEM) 97. They are working on solving the structure. Sigma receptors were discovered in the 1970s because of their response to analogs of drugs that activate opioid receptors. In 2016, the Kruse lab determined the structure of Sigma 1, an unusual receptor that cross reacts with many drugs and has been linked to neurodegenerative diseases, addiction and pain.
In a newer technique, they are using evolutionary analysis and prediction to solve structures from scratch, a collaboration with Harvard systems biologist Debora Marks. Structural biologists need a starting point to determine a new atomic structure, but there was no homologous protein for RodA, a transmembrane protein that bacteria use to build their cell walls. Evolutionary sequence data has been used to predict protein folds, but never to solve a crystal structure of a protein of this size, Kruse says. In this case, they generated hundreds of possible templates and tested them all. A few models were good enough to solve the structure (see image), published online 28 March in Nature. For the computational structure approach, they relied on molecular replacement software implemented by SBGrid, generating a shell script to iteratively run hundreds of searches.
Radical Reactions - Yvain Nicolet - Institut de Biologie Structurale
Sharing some of our #SBGrid member tales from the last year. This one from January 2023.
The periodic table may be an icon of chemistry, but a cluster of elements at its center gives biology some essential pop and sparkle. Yvain Nicolet at the Institut de Biologie Structurale (IBS) in Grenoble, France, wants to learn exactly how those transition metals in the middle of the table put more pizzazz in proteins.
About one-third of proteins contain transition metals, such as cobalt, copper, molybdenum, or iron. The Nicolet lab mainly studies metalloproteins that contain iron-sulfur clusters but also scrutinizes enzymes that make these clusters. "Transition metals are often key components of the proteins in terms of function," Nicolet says. "They are only few atoms in a structure and sit in specific sites, but they enable specific activities, notably in enzymes, but not only in enzymes." Metals give proteins some superpowers in the form of new chemical properties and functions not otherwise possible with combinations of the 22 amino acids.
"They enhance the reactivity of the proteins by making many strange active sites that are very interesting," he says. These reactions can be blazingly fast with complex chemistry. And they are technically challenging to study.
The lab investigates enzymes that use metals, as well as proteins that build the metal cofactors and insert them into the enzymes. "We try to trigger the reaction in a crystal and to trap the different intermediates during the reaction to characterize by structural biology," Nicolet says.
Many metalloproteins evolved in a time before oxygen and can only make their razzle-dazzle moves in an anaerobic space. To prevent oxygen from destroying their molecules, experiments in the Nicolet lab take place in a series of six large anaerobic chambers filled with nitrogen.
The researchers reach in through gloves built into the airtight boxes to work with samples and equipment. There, cells are grown and broken up. The cell extracts are cleared and proteins separated by chromatography. Crystallization robots in one glove box screen for optimum conditions that researchers can manually reproduce in another box and freeze in liquid nitrogen. From there, the synchrotron is only 500 meters away.
"We determine 3D structures at moderate to high resolution," Nicolet says. "At first, we aim at determining where the metals are in a given protein, and how they interact with their environment (the protein matrix). We also aim at determining the structure of the metallocofactors or metal binding sites in terms of ligands and geometry.
"Then in a second step, we aim at understanding how these metals confer a specific activity to the protein," he continues. "For instance, in an enzyme, we would get intermediate states with the metal in a different coordinate and/or redox state to better understand what is going on during the reaction."
One milestone of the lab's work arose from successfully trapping an unexpected radical intermediate of NosL, a radical S-adenosyl-L-methionine (SAM) enzyme, in the process of generating a peptide with potent activity against gram-positive bacterial pathogens (Science, 2016).
Crystallography provided the position of the atoms, and for this paper they also used a spectroscopy technique called electron paramagnetic resonance (EPR), which gives the position of unpaired electrons near atoms.
Two months later, the group reported results from a study in which they carried out radical based chemistry catalyzed by the radical SAM enzyme HydE from beginning to end in a crystal (Nature Chemistry, 2016).
The image shows substrate processing and product transfer within HydE cavity during the FeFe-hydrogenase active site assembly process. Credit: Y.Nicolet
More recently, Nicolet's team characterized intermediates in the assembly of the nitrogenase, a key player in the global nitrogen cycle. The enzymatic complex nitrogenase catalyzes the reduction of nitrogen gas N2 to ammonia NH3, transforming nitrogen and returning it to plants. Specifically, the paper reported the X-ray structure of NifB protein from a nitrogen-fixing bacteria, one of a dozen proteins required to assemble the nitrogenase active site (Journal of the American Chemical Society, May 2020).
"For nitrogenase, there is a dedicated machinery with very, very challenging chemistry involved, and this is what we are attacking over the few next years," he says.
Nicolet was born and raised in Grenoble, France, where he now lives and works. Growing up in the city, he became an accomplished cello player with ambitions of becoming a professional musician. He started college in Grenoble. When a music school in Paris turned down his application, he chose to pursue science at what is now the University of Strasbourg. In Strasbourg, all the science classes emphasized the three-dimensional structures of molecules, whether it was enzymology, DNA replication, or proteins. He completed his undergraduate degree and stayed for graduate training in chemistry, biology, crystallography and nuclear magnetic resonance (NMR).
In a dinner discussion, a friend noted his interest in inorganic chemistry and suggested a structural biology lab in Grenoble working on metalloproteins. He moved back to complete his PhD at what is now the Université Grenoble Alpes. Nicolet worked on the structure of the [FeFe]-hydrogenase (an enzyme that makes or use molecular hydrogen), basic science with a potential application in producing alternative fuel.
From there, he joined the Drennan Lab at the Massachusetts Institute of Technology. In Cambridge, he discovered radical-based chemistry working on this newfound Radical SAM Proteins superfamily, now known for their ability to synthesize many vitamins, cofactors, or antibiotics.
He moved back to Grenoble for a one-year postdoctoral fellowship with the European Synchrotron Radiation Facility. In 2004, he joined IBS as a scientist and became a group leader there in 2016.
The Nicolet lab often dives into the molecular mechanisms of phenomenon discovered by other groups. "In my lab, we are not at the beginning of the discovery, and we are probably not at the end of the process," he says. "We focus on the molecular mechanisms inside the enzymes."
In a new challenge, the lab has set up a system to use cryo-electron microscopy to examine the assembly dynamics and reactions at room temperature without oxygen. Nicolet is interested in the basic science of radical chemistry, but he also hopes to be able to harness a radical enzyme and modify its catalytic activity to produce a molecule through directed evolution, such as a better antibiotic.
Nicolet also enjoys taking a bigger picture view of the nitrogen cycle, thanks to his garden. He came late to the activity and confesses that he is not good at growing vegetables. "You can observe the nature or the interaction between the different organisms that live together from bacteria to insects and plants," he says. "For me, it's the best way to contemplate what Darwin described as natural selection. And evolution. I'm not very good at producing vegetables, but I really enjoy seeing how all these organisms compete together or help each other as well.
Ramaswamy Subramanian
Institute for Stem Cell Biology and Regenerative Medicine
One day, a recently retired colleague visited S. Ramaswamy in his University of Iowa laboratory and presented him with a fish from a Canadian lake and a question. Why was the fish blue? Normally, North American walleyes are golden yellow, but more seem to be turning blue in summer months.
The unexpected question hooked Ramaswamy, who goes by Rams, even though it had nothing to do with his core research program. But he places a high value on curiosity-driven research. “That has been the key to a lot of the work in the lab,” says Rams, whose most famous extracurricular research probed embryonic cockroach milk crystals (more on that later).
In the case of the curiously blue walleye, the Rams lab embraced the project with structural and spectral experiments on the blue pigment protein complex. They documented a small new fluorescent protein (sandercyanin) with unique far-red reflective properties. In the fish, the outer pigment layer may act as a protective sunscreen to absorb excess damaging UV radiation let in by the Arctic ozone hole, reported the Rams team and their collaborators in 2016 in PNAS.
Rams grew up in Southern India. He started his scientific career there as a physicist, with a masters degree in electronics. Like the fish, structural biology appeared to him out of the blue. In this case, it was a lecture on protein crystallography in the 1980s. The idea that the position of atoms could explain how proteins work made sense to him. After all, that’s how computer chips were designed (atom by atom). And only a handful of structures had been determined, making it a field with great potential.
In another bit of luck, he found a PhD supervisor setting up his own lab in India after being freshly trained by Purdue University structural biologist Michael Rossmann. After his PhD at the Indian Institute of Science in Bangalore in 1992, Rams left for a postdoctoral fellowship at the Swedish University of Agricultural Sciences and stayed on as a researcher in Uppsala.
A collaboration with University of Iowa (UI) turned into an invitation to set up an X-ray crystallography program there. In 2000, Rams moved to Iowa. Over the next 10 years, he added nuclear magnetic resonance (NMR) and electron microscopy facilities—and acquired a new last name, in the tradition of earlier generations of new U.S. citizens.
He had been known as S. Ramaswamy, with S. standing in for the first initial of his father’s last name and Ramaswamy, his given name, serving as the reference name in journal author credits. But to become a U.S. citizen, “S” was rejected as a stand-alone name. Expediently, he expanded it to Subramanian and made it his new surname for legal purposes.
In 2009, Rams returned to Bangalore to set up the Institute for Stem Cell Biology and Regenerative Medicine (InStem). He has maintained his faculty position and collaborations in Iowa (traveling back several times a year at first). In India, he continued his structural biology research, setting up a suite of tools and facilities, including X-ray, NMR and cryo-EM.
In Bangalore, he also co-founded the Center for Cellular and Molecular Platforms (C-CAMP) and served as chief executive officer until 2016. India’s first large biotechnology incubator, the center paired start-up companies with the problem-solving abilities of a strong academic institution. A new idea there at the time, Rams says, the center supported 50 companies by the time he left the helm.
In May 2019, Rams will move back to the United States, this time to Indiana to direct the Bindley Bioscience Center at Purdue. There, he will be answering the question, “What are new fun things one can do at the intersection of biology and data science and engineering?” His structural biology lab will be moving more into cryo-electron microscopy to elucidate enzyme activity in larger protein complexes.
“One thing that makes structural biology special is the dramatically collaborative way by which methods have been developed and software not only developed but made available and integrated,” Rams says. “It’s very special about this particular community. It’s been an important part of my scientific work.”
The core research in the Rams lab revolves around the exquisite details of how enzymes transfer electrons that enable oxygen’s fundamental role. His research in mechanistic enzymology of non-heme metal enzymes explores how iron and other metals catalyze reactions outside of the well-known oxygen-carrying iron atom in hemoglobin.
“When [life on earth] went from being anaerobic to aerobic, the trick was to utilize oxygen,” says Rams. “It’s all about electrons. It’s all connected to that one or two electrons going from iron to oxygen and making oxygen reactive to do the things that chemistry cannot do otherwise. This was a primary switch between anaerobic and aerobic.”
Early, his lab determined crystal structures of dehydrogenases, a liver enzyme that begins to detoxify alcohol in the body. In Iowa, he became interested in infectious diseases. In 2003 in Science, his lab reported a crystal structure of a new way that nonheme iron enzymes activate oxygen in the enzyme naphthalene 1,2 dioxygenase. They showed how the enzyme manages the critical step of in catalyzing the stereo-specific cis-dihydroxylation reaction consistently and correctly. (Stereo specific refers to selecting one molecule over its mirror image, also known as handedness.) Another program advances our understanding of the molecular mechanisms of sialic acid uptake by gram-negative bacteria and help lay a foundation for finding new targets to overcome antimicrobial resistance.
Since moving to India, he started work on nucleotide sugar transporters, proteins in cells that move sugars from the cytosol to the endoplasmic reticulum and golgi in cells. His team raced to determine the first structure, but they were scooped by another group. They continue to work on it, “as there is a lot more to be done to understand on how these transporters work,” Rams says. The work is a collaboration with the laboratory of Jeff Abramson at University of California, Los Angeles, a former colleague of Rams at Uppsala.
Above: These structures show the molecules in bacteria that scavenge 9-carbon sugars and use them in metabolism. In this project, the Rams lab collaborates with Rosmarie Friemann at Gothenburg University and Ren Dobson in New Zealand.
Meanwhile, another curiosity-driven project had skittered into the Rams lab. In 2006, the UI campus hosted the largest collection of the world’s cockroaches. The collection included the Pacific Beetle cockroach, commonly found in Hawaii and the only species known to give birth to live young. Small crystals in the gut caught the attention of an undergraduate student in the Rams lab, who also worked in the cockroach lab of Barbara Stay. He ran them through the X-ray beam and discovered they were protein crystals.
The tiny crystals eluded several early attempts to determine their structure using X-rays and NMR, but eventually the Rams lab and their collaborators determined the structure of the cockroach milk crystals. Pound for pound, the milk crystals have times the energy of the same mass of dairy milk. In the mother cockroach, the concentrated milk crystals nourish 9-12 developing embryos.
“It took us 10 years, but this structure was a feat,” Rams says. The team used single-wavelength anomalous dispersion (SAD) to gather data on the mixed protein crystals.
After they determined the structure, the Rams lab synthesized the gene and grew the protein crystals in yeast for further study. “This could be an excellent source of nature-inspired high-energy food,” he says.
The study, published in the July 2016 journal for the International Union of Crystallography, is believed to be the first report of a detailed structure from a crystal naturally grown in vivo rather than in vitro from overexpressed proteins. But it won’t be the last. Rams says a new area of research, in vivo crystallography, has emerged to study nature’s crystals, a tool functions in health and also a byproduct of some disease processes.
In fruit flies, for example, certain protein crystals found in the blood cells at a specific stage of development allow the embryos to survive. In another interesting example, ”nature has used crystallization as a mechanism to help protect and store highly concentrated proteins in seeds, and this has been well documented,” Rams says. “But, how these crystals are packed and what makes these proteins crystallize in a very heterogenous environment is yet to be understood.”
Rams is a foodie who likes to cook and eat the local vegetarian cuisine wherever he lives, but he hasn’t tried the milk crystals, either from cockroaches or yeast. However, he jokes that if someone made a high-protein beer from those yeast, he would consider tasting it.
As a physics undergraduate student in Munich, Gerhard Wagner worked on an esoteric atomic measurement of iron in a protein molecule. Then he heard from his supervisor, who was on sabbatical at Bell Labs in New Jersey. There, the same molecule, hemoglobin, the iron-rich protein that carries oxygen in red blood cells, was being probed by nuclear magnetic resonance (NMR) spectroscopy.
The technology caught Wagner’s attention. Why measure a single parameter, as he was doing, when you could measure many aspects at once with NMR and learn so much more, he asked himself. Little did he know, but that thought launched a lifetime of NMR research.
Wagner hadn’t planned on a career in academia. He was born in Czechoslovakia just as World War II ended. His German-speaking family became refugees, fleeing to nearby Bavaria and starting again with nothing. In science, Wagner also helped develop a new field from scratch.
When he graduated from Technical University in 1972, he found a sweet spot in Switzerland—home of his girlfriend, skiing, and a PhD training position with Kurt Wüthrich, one of the early scientists to use NMR on proteins and who later won a Nobel Prize. He spent most of the next 14 years in Zurich.
At ETH Zurich, Wagner launched a family and a career in NMR that soon brought him to the attention of the world’s top structural biologists. At the time, protein structures could only be determined by X-ray crystallography. Wagner helped develop suites of techniques for the fledgling NMR analyses of proteins that made it possible to assign resonances and solve their structures.
Early on, he observed one of the NMR advantages: protein dynamics. “I found a lot of mobility in proteins,” he says. These days, NMR is well known for its unique ability to reveal thermodynamic and kinetic aspects of proteins. More than 40 years ago, those protein wiggles were nearly impossible for some to imagine.
Wagner was testing the new techniques on a cow protein with stable ring-shaped parts (called aromatic side chains in chemistry parlance). Analyzed by NMR, the rings flipped when the temperature went up or down, opening the protein structures by several Ångströms. The unexpected results were controversial.
Wüthrich planned to talk about the new data at a physics conference in Germany. Behind the stage, conference organizer and Nobel Laureate Max Perutz didn’t believe the results and objected to the presentation. Perutz famously had solved the structure of hemoglobin, which also sported aromatic side chains. Another prominent scientist, Robert Huber, who later also was awarded a Nobel Prize convinced Perutz to let the talk proceed as planned, Wagner says. (Later, the same data provided a test bed for the development of molecular dynamic simulations by Martin Karplus.)
After a one-year postdoctoral position at the Massachusetts Institute of Technology in Cambridge, Wagner continued his work in Wüthrich’s lab in a postgraduate teaching and research position called privatdozent. Soon it came time to present Wagner’s latest work, the NMR structure of cadmium-bound metallothionein, a small protein that binds zinc or other metals in plants and animals. Wagner got a worried call in the middle of the night from for Wüthrich. It seems the NMR structure was very different from a crystal structure of the same molecule being presented at the same meeting. Wagner and his coworkers had applied a new technique, proton cadmium correlations, to determine the topology and structure. He scrambled to review the data. It all checked out correct.
“It was also an early time in crystallography, so people could still make mistakes,” Wagner says. “It gave me some visibility. It showed NMR could do a structure and get it right.” Later, the two NMR and crystal teams published a joint paper showing agreement between their final metallothionein structures.
NMR eventually became an accepted method for solving protein structures, especially of smaller proteins in lower concentrations. Wagner took a faculty position at University of Michigan in 1987. There he just had time to develop the first triple resonance experiments, a strategy that is now the basis for most protein NMR, before he was recruited to Harvard Medical School in Boston in 1990.
“When I came to Harvard 25 years ago, I came as an expert in NMR,” Wagner says. “I thought I should also have an important biological project.” He looked for areas with little research and landed on translation initiations, or how a transcribed gene is turned into a protein.
After a gene is transcribed into RNA, a large protein complex grabs hold of the messenger RNA tail to prepare to make a protein. In a 2003 Cell paper, Wagner’s lab reported the structure of the first two proteins in this complex (eIF4E and eIF4G) and how they enable the ribosome to bind to the 5’ end of mRNA and start making protein.
Above: In this NMR image, a complex of two proteins (eIF4E in red and yellow and eIF4GI in blue) attach to the 5’ cap structure of mRNA, and eIF4G forms a “molecular bracelet” around the N-terminus of eIF4E, prompting the initial event of ribosome recruitment to start making a protein. Courtesy G.Wagner
“Then the idea came that oncoproteins have long 5’ UTRs,” or untranslated tails, Wagner said. “Our hypothesis is that nature developed long tails to make it more difficult to translate dangerous proteins.” Other labs have shown that the long tail effectively down regulates some proteins. In cancer, such controls on the oncoproteins may be broken, he says. Restoring them may be a new way to treat cancer.
Wagner and his collaborators screened for small molecules to inhibit some of the translation initiation proteins his lab had characterized. Now they have a major program to improve the compounds they found with anti-cancer activity.
These days, NMR remains well suited for characterizing smaller proteins and to catch the gymnastic moves of molecules in their active biological roles. In particular, Wagner believes NMR has potential to illuminate RNA in complex with other proteins. “Another area where NMR may have some impact is proteins that are only partially folded,” he says.
For example, the nuclear factor of resting T cells has a long unstructured tail hanging in the cytoplasm of cells. The tail has many phosphorylation sites, keeping it in a resting state. Working with another DFCI collaborator, Wagner’s group developed a new NMR technique (known as direct 15N detection) to elucidate a key internal step in activating T cells, which is important to fight infections but also needs to be regulated to prevent rejections of organ transplants. In a collaboration with Haribabu Arthanari at Dana-Farber Cancer Institute, the team identified a key molecular interaction in activated T cells. The Wagner lab is developing small molecules that inhibit the interaction, ideally targeting a protein-protein interaction with fewer side effects than current anti-rejection drugs.
In another project, Wagner’s group has found a better way to study membrane proteins in a more natural environment. A team led by postdoctoral fellow Mahmoud Nasr improved the design of tiny nanodiscs, a popular model of cell membranes used to study proteins entering cells.
The Wagner lab’s modification of the design made the nanodiscs more stable using some fancy chemistry bonding membrane scaffolding proteins. The resulting covalently circularized nanodiscs can be precisely sized from 9 to 50 nanometers and used to study viruses and other membrane interactions by adding a receptor. In their first published demonstration in 2017 in Nature Methods, the team and its collaborators observed polioviruses opening a putative pore and injecting genetic material through the nanodiscs, as the virus might do when it infects a cell.
Left: A cartoon illustrates the poliovirus (ball) attaching to its receptor CD155 in the membrane interior of a DNA-corralled nanodisc. Right: The EM micrograph series shows the sequence of the poliovirus engaging with the membrane, ejecting RNA through the nanodisc. Then the empty virus leaves behind a putative ejection pore.
When Wagner moved on to study HIV and the process by which it fuses with a cell membrane, the nanodiscs became too big for NMR. The ongoing work requires cryo-EM analysis. Collaborating with William Shih’s lab at the Wyss Institute, Wagner’s group developed a larger nanodisc design with the lipid bilayer inside DNA scaffolding. He sees many possibilities ahead with the new technology, including nanodisc complexes.
Gaya Amarasinghe
Washington University in St. Louis
On a recent visit to the laboratory where he worked as an undergraduate, Gaya Amarasinghe thought about his scientific journey from student to professor. At first glance, his research interests seem to have drastically changed. Back then, he was studying signaling in cancer biology, and how oncogenes communicate within cells. Now, his group at Washington University in St. Louis investigates how some of the world’s deadliest viruses, such as Ebola, outcompete the host and cause disease.
Yet, he decided, not much has changed after all. “In the last 20 years, I haven’t done anything differently,” joked Amarasinghe, who trained as a structural biologist and biochemist. “My group now studies proteins from viruses and bacteria, but they interact with and sometimes hijack the same immune signaling cascades I studied before. I’m asking the same questions.”
Amarasinghe’s group examines how the host immune system recognizes foreign pathogens and how normal immune signaling changes in the presence of microbes.
“We ask simple questions, and we answer them using simple experiments,” Amarasinghe says. “What are the initial components in the system, what does the structure look like, and what are the dynamics of these molecules? Then we ask what happens to those properties when they interact with a virus. What does that interaction look like? How does it move? What changes?”
It was a question that first made them curious about Ebola and related Marburg viruses: How do they evade the pathogen detection system that the immune system uses to distinguish self from non-self?
Most people know these filoviruses as causes of severe, often fatal, outbreaks of hemorrhagic illness, usually in Africa, where people first catch the contagious diseases from animals.
Few viruses are as dependent upon first interactions with the host as Ebola and its cousins. Unlike other viruses with multiple ways of manipulating an immune response during an infection, Ebola and Marburg viruses begin with a knock-out punch to the immune system defenses. It happens at the earliest stage, Amarasinghe says.
The critical moment comes when the innate immune cells that function as sentinel cells in the host, such as dendritic cells and macrophages, recognize the viruses as unwanted foreign invaders—or not, as is often the case.
A key player is a crucial viral protein called VP35. Without a working VP35, the viruses cannot defend early immune responses during infection. It also blocks many steps of the host’s innate immune response, including one that ramps up the antiviral immune fight.
The first attempts to answer the question about initial recognition of the virus were anything but simple. “The hardest part was getting the material we needed,” Amarasinghe says. It was a brute force effort to purify the protein. He estimates it took the equivalent of 10-20 people-years of work in many groups, including his own, to finally isolate well-behaved VP35 by genetic recombination techniques.
In 2009, his group published the first of a half-dozen papers, which include at least 10 different VP35 structures of what the interferon inhibitory domain (or IID) from several Ebola strains and Marburg, which have different disease courses.
“VP35 IID was nothing like anyone predicted,” Amarasinghe says. “This protein is hiding the non-self signal that would otherwise get recognized by the immune system. It’s hiding the signal in plain sight.” The protein doesn’t even need to move much. It has a fold that fits neatly over the non-self signal, an end of the detection RNA found in every cell.
The group also found a fundamental difference between Ebola and Marburg viruses in how they recognize the host RNA and hide it. “They are equally virulent and pathogenic, but there is a key difference at an early stage,” he says. “Why does that matter? What happens in early stages determines the outcome of infections.” For example, Reston Ebola virus, a close cousin of the more virulent filoviruses also uses VP35 to bind RNA, but differences in other parts of the virus replication cycle make Reston non-pathogenic in humans.
Once they knew what the protein looked like in the related hemorrhagic viruses, Amarasinghe’s team helped design mutated versions to model the disease and better define the biology. The mutated viruses were immunogenic.
“Very cool, because now we have virulent and avirulent near-isogenic viruses to study the disease in animal models” he says. This knowledge can be used to develop actual vaccine candidates with different molecular backbones or using smaller subunits for safety, he says.
Most of the lab’s work happens in test tubes with non-infectious viral proteins. “We have collaborators working in biosafety level 4 that put some of these crazy ideas to work,” he says. Amarasinghe and his collaborators are testing the biological mechanisms in animal models.
A second set of discoveries from their work helps explain why Ebola and Marburg are not responsive to interferon, a potential treatment given for many viral infections, but as it turns out it does not work well in filoviral infections. VP24, another protein encoded by the Ebola virus binds to a key cargo transporter called karyopherin, which is responsible for moving important signals, or transcription factors, into the nucleus to trigger an immune response. Viruses are known to block dendritic cells from maturing, preventing the normal interferon signaling necessary for an adaptive immune response. Amarasinghe and his colleagues have shown that VP24 also inhibits molecular signaling from therapeutically delivered interferon.
“Typically we think of a host-microbe arms race, with the host evolving away from the pathogen,” Amarasinghe says. “In this case, the place where the viral protein binds is unable to evolve away, because the host would become non responsive to its own immune signal. The virus identified an area where mutation change rate is low.”
Most of the lab projects fall into one of two categories—how host factors recognize the signature or foreign pathogens, and how host signaling changes in the presence of a microbe.
The host stress response to a virus, and not the virus itself, could be responsible for some diseases and point the way to better treatments. This could be the case with the human respiratory syncytial virus (hRSV), an infection most kids acquire by age 2. It can be fatal to infants and the elderly. It is in the same family as another emerging infectious disease people catch from animals is Nipah virus, an often fatal infection that causes encephalitis and severe respiratory distress.
Amarasinghe and his colleagues purified and solved the first crystal structure of an hRSV protein known as NS1. They reported their findings in 2017 in Nature Microbiology. They found a duplicated structural fold that suggests NS1 may have a greater role in regulating host responses than previously appreciated. “It’s not quite falling off log, but it’s close,” he says about the functional revelations.
In the future, Amarasinghe says his lab will continue to explore what drives infection in the early stages from host response point of view, aiming to understand factors that determine outcome in longer term persistent infections. The knowledge may be useful in other bacterial and viral systems, including the effect of co-infections on outcomes. “None of these exist in isolation,” he says.
Amarasinghe starts many days as early as 4:00 am and most mornings with a long run or a bike ride. It keeps him in shape for occasional long-distance mountain biking or skiing.
By his name alone, Amarasinghe may have been destined to work with proteins and RNA. Every letter in his full name represents an amino acid. He pointed out that his first name, GAYA, also forms a tetraloop (guanine-adenine-pYrimidine-adenine).
Orsolya Barabas
European Molecular Biology Laboratory, Heidelberg
Thickly forested slopes define the environs around Heidelberg, Germany, the headquarters of the European Molecular Biology Laboratory. In her hillside EMBL laboratory, Orsolya Barabas probes the small pieces of moveable DNA that define the landscape of modern genomes.
Surprisingly few people have heard of transposons. These “jumping genes” have been flitting around genomes for millions of years, changing locations or introducing new copies of themselves all over the place in plants and animals. They escaped notice until the 1940s, when Barbara McClintock observed them in maize, where they dominate the DNA, eventually earning her a Nobel Prize in 1983.
At the last best guess, about half of the human genome is composed of mostly dormant remnants of transposons. Bacteria, on the other hand, rely on highly active transposons to share genes to help them adapt and survive, such as those that confer antibiotic resistance.
“Transposons are very simple DNA vehicles that can help integrate genetic cargo into the genome,” says Barabas, who works both sides of the potential medical applications, starting from the atomic details. Her group studies transposons used in genetic engineering, seeking to make their gene delivery more efficient. On the flip side, she also scrutinizes transposons that spread antibiotic resistance genes among bacteria, hoping to limit that activity.
The scientific world has been buzzing about CRISPR/Cas9 for targeting and editing a specific location on DNA, but transposons have the edge when it comes to the step of inserting a gene, Barabas says. Transposons also have some advantages over viral vectors, a more established way to insert genes efficiently.
“Transposons are the first line of non-viral gene delivery tools,” she says. “Their applicability is greatest for ex vivo cell engineering.” The early use of transposons has mostly been in personalized immunotherapy clinical trials, in which a patient’s immune cells are withdrawn, genetically altered to recognize and kill cancer cells, and then injected back into the patient.
Barabas and her colleagues have found a way to improve the first and most popular such tool. Called Sleeping Beauty, the transposon was synthesized about two decades ago by Hungarian researchers. The awakened transposon was reconstructed from ancient DNA sequences estimated to be more than 10 million years old, which had been fished out of the genomes of trout and salmon. In their lab in Germany, these same researchers have since modified Sleeping Beauty to be 100 times more efficient, a version called SB100X.
Above: The first structure of the transposase protein of the Sleeping Beauty transposon is modeled into full transposition machinery. Image Franka Voigt & Irma Querques. Courtesy Nature.
“The coolness of this transposon is that there is no active copy in any genome,” says Barabas, who set up a collaboration with the original Sleeping Beauty team. “It’s a rational reconstitution of an evolutionary pathway. From an application point of view, this is cool because you can use the tool in any genome without unwanted mobilization of genomic DNA.”
In a 2016 paper in Nature Communications, Barabas and her co-authors reported the first crystal structure of the key piece, the SB100X transposase enzyme. Transposases are the workhorses of transposons, clamping, cleaving, and rejoining the DNA.
Using the new structure, the team tweaked some amino acids on the surface of the enzyme to make versions that could bind even better to the DNA where genetic integration occurs.
“They were the first rationally designed hyperactive transposon variants,” she says. The new design has a 30 percent efficiency boost. These variants are not yet in cancer trials, but for patients waiting for cancer treatment, Barabas estimates they may shave as much as a week off a month-long wait for SB100X to supercharge their immune cells.
“As well adapted parasites, transposase enzymes are not naturally optimized to work efficiently,” she adds. “This leaves a lot of room for optimizing their activities for human use or genome manufacturing at will.”
One limitation of transposons for genetic engineering is that they insert cargo in many different places on the DNA. In contrast, the CRISPR/Cas9 system can target a specific DNA site, but then it relies on the host’s DNA repair mechanism to insert a gene, which isn’t good enough for a medical setting, she says.
“Ideally, it would be nice to combine these things: Site specificity and integration,” Barabas says. Until that vision becomes reality, her lab continues to develop more hyperactive variants and seek ways to target them better.
A different project in the lab has the opposite goal: To derail transposon activity—at least those that spread antibiotic resistance among bacteria. The numbers of microbes resistant to multiple antibiotics has been increasing globally, causing concern among scientists and doctors about the threat to public health.
“The fact is that resistance genes already exist for all antibiotics we use,” she says. “There is not a single antibiotic for which a resistance mechanism does not exist.”
Transposons largely drive the spread of antibiotic resistant genes among bacteria, even within the resident microbes in our gut. Stress—such as antibiotic treatment—can trigger bacterial transposons to amplify.
The Barabas lab started with conjugative transposons, which move between the single-cell microbes during the process scientists jokingly refer to as bacterial sex. Normally, two single-celled microbes meet and form a membrane tube to swap DNA and proteins, including transposons carrying genes that confer drug-resistance.
They solved the first structure of a transposon of this class. A 2018 Cellpaper reported the transposon DNA in conjunction with the protein machinery that plucks out resistance genes. The structure shows how the enzyme can peel open the DNA double helix in two parts. A single stranded segment forms a bubble that can touch down almost anywhere else without needing to match a specific sequence.
Above: This structure captures a pair of transposases (two shades of blue) with their DNA cargo (red and black) after the transposon jumped out of one genome. The pink protein segments are poised to unpeel the DNA when the transposon lands on another target genome. This conformation reveals an unexpected stability that may help develop new strategies to prevent the spread of antibiotic resistance. Courtesy O. Barabas. Courtesy Cell.
Based on the structure, Barabas and her colleagues predicted two types of molecular inhibitors. One peptide blocks the transposase protein from moving to its activated conformation. The second is a small DNA and binds to the open site within the transposon, blocking the DNA strand replacement needed for resistance transfer.
She envisions potential inhibitors may be used in combination with a course of antibiotics, to prevent resistance spreading.
Barabas grew up in Hungary, the daughter of two chemical engineers. She remembers being fascinated by how things in everyday life could be explained by chemistry and other phenomena people can’t normally see. When it came time to train for a career, Barabas decided to direct her imagination and creativity toward chemistry and basic research. Her father was concerned, reasoning that the country could not support many basic researchers.
At Eötvös Loránd University in Budapest, Barabas studied structural chemistry. For her masters, she crystalized small molecules used in drugs and pills, learning how their structure influences their actions and effects. Then she moved on to enzymes, looking at their effect on nucleic acid metabolism and maintenance. When she finished her PhD in 2005, she was riveted by how proteins juggle with DNA.
She crossed the Atlantic Ocean for a postdoctoral position at the National Institutes of Health in Bethesda, Maryland, USA and was introduced to transposons in the lab of Frederick Dyda. “I had no idea what transposons were, but they sounded fascinating—dedicated elements in the genome that fancy to hop around, shaping us into who we are,” Barabas says.
She became a group leader at EMBL in 2009. Over time, Barabas has become increasingly intent on connecting basic structural knowledge to applications.
“It’s important that we really use the structure to understand its function, and also develop it into something useful for society,” she says. “For me, the applicability of the acquired knowledge is critically important. Taxpayers pay us to have the most thrilling job, and I need to pay them back.” To help push past the basic discoveries, the Barabas lab does biochemistry, biophysics, structures, cell biology and other methods.
Collaborations abound. To explore how the antibiotic slaughter of gut bacteria affects their susceptibility to antibiotic resistance genes, she has teamed up with computational geneticists and a mouse biology lab, as well as with a clinician for human stool samples. This will allow investigating how transposons spread in microbial communities in the gut and what triggers induce the movements. More structures are on the menu to compare mechanisms among the many different transposons.
Like others in her sporty college town, Karolin Luger heeds the call of the mountains she can see from her laboratory at University of Colorado Boulder. Trail running and a daily 20-mile round-trip bike commute at altitude have kept her fit, but she would like to put to rest the rumors that she runs her research collaborators ragged on strenuous hikes. “All these crazy stories,” Luger says. “I only did that once.” And lately, she says, she’s the one who has a hard time keeping up on group outings.
Her collaborators can relax, but Luger wants to see more dynamic motion in the chromosome structures she studies. After all, they have to move to do what they do. Luger’s research addresses a fundamental question: How does the human genome store information and then access that information at the appropriate time? Part of the answer, she and her collaborators have learned, goes back billions of years.
In animals, a full genome must be squeezed into the nucleus of each cell. In humans, that means six feet of DNA must fit into something less than one-fifth the size of what the human eye can see. This can happen thanks to the compact packaging of DNA into an assembly called chromatin. Chromatin consists of a long strand of tightly twisted DNA wrapped twice around repeating flat spools of histone proteins, like a string of beads. The protein-DNA spools, or nucleosomes, are the basic repeating unit of chromatin. They are further folded and arranged into a higher order structure that Luger’s lab also studies.
“Imagine a super-long sewing thread and wrap it around a million tiny hockey pucks,” Luger says. “It’s actually really a miracle that there are not knots and tangles.”
Chromatin controls access to genes in ways Luger and her colleagues are still learning. The tightly packed hockey pucks, or nucleosomes, must unspool to activate certain working genes, to duplicate DNA during cell division, or to repair damaged DNA, and then rewrap. “We’re still far away from figuring out how that works,” she says.
Complicating matters, genes can be independently activated or silenced by small chemicals that tag the histone spools or the DNA itself. Luger’s lab is trying to figure out how those small epigenetic tags affect the packaging of the hockey pucks, especially during development, as the same genetic blueprint produces such different cells—brain, heart, muscle, liver and skin, blood, bone.
Luger grew up Austria in a landscape dominated by mountains and science. Her father was an engineer, and her brothers were interested in electronics and physics. Luger was first drawn to botany and zoology. She studied microbiology at University of Innsbruck. Bleak job prospects and a rewarding undergraduate experience in a biochemistry lab inspired her to pursue a PhD in protein engineering at University of Basel, Switzerland.
Given a free hand by her mentor, she wanted to learn if a protein could fold normally if it came out of the protein-translating ribosome backwards, somewhat like a breech birth. (The answer is yes, it can.) She developed a new technique to test the idea, joining two ends of a gene together and cutting it open in a different place. The results were published in Science in 1989. Her studies with such proteins ended with her doctorate, but others have used the information in evolutionary studies to detect genes that make proteins of similar structure and function, but whose gene sequence might be permuted in this manner.
Her PhD program had required her to take classes in structural biology. To satisfy a new craving to learn crystallography, she joined the lab of Timothy Richmond at Swiss Federal Institute of Technology as a postdoctoral fellow. Her project resulted in the long-sought high-resolution structure of a nucleosome at 2.8 angstrom, published in Nature in 1997.
“I had no idea what a super-gnarly problem it was,” she says. “I learned the hard way, but it was a good learning experience.” She also learned that she had a visual brain highly suited to structural biology and developed the methodology (now used in many labs world wide) to make nucleosomes for crystallization and for biochemical and biophysical studies.
She joined the Colorado State University (CSU) faculty in 1999 and became a Howard Hughes Medical Institute investigator six years later. In 2015, she moved her lab to the University of Colorado, Boulder. Luger continues to collaborate with CSU faculty at the Institute for Genome Architecture and Function, a research consortium she and two fellow CSU women faculty members founded in 2015.
Her lab’s work has broadened from solving crystal structures to using cryo-EM, and also to asking scientific questions using structural biology as one of many tools, including live cell experiments. Luger likes to find new ways to see something that wasn’t previously observable. “I almost like the process of doing science more than the actual results,” she says. “I love the process of discovery, to figure out mechanisms and trick the system to tell me its secrets.”
She has also become more action-oriented. “We can stare endlessly at structures,” says Luger, who admits she still is blown away by the transformative power of hydrogen bonds and amino acid packing. “But we need to think about function even more. Most of the structures we study are machines and have to move. We are more and more interested in catching these machines in the act of going about their business. It’s immeasurably harder than looking at the static structure, which is already hard enough.”
Luger describes the narrative arc of her lab’s research path as a “directed random walk.” She adds, “My feelers are open all the time to see what I can learn from others. I just love collaborating with people and integrating ideas out of left field into my research program. It drives people in my lab crazy sometimes, because they are the ones that have to do the hard work.”
In one collaboration reported in Science in 2006, Luger and her colleagues examined a crucial binding point between a protein on the virus that causes Kaposi’s sarcoma and a pair of histone proteins in the nucleosome. “This was the first time it was shown how nucleosomes can serve as docking stations for other proteins,” Luger says. “As far as the virus goes, this gives its genome a nifty way to hitchhike on the host’s chromosomes to escape its defense mechanisms.”
Two years later, Luger and her collaborators used new techniques to mimic the chemical marks on tightly wound DNA-protein complexes that silence gene expression. They reported that repressive epigenetic marks on histone proteins cause nucleosome arrays to become more compacted, while activating methylation did little to the chromatin. The findings were published in Nature Structural & Molecular Biology.
Scientists have wondered about the origins of nucleosomes. Luger and her collaborators looked for answers in archaea, an extended family of ubiquitous single cell organisms thought to be the ancient precursors of eukarya. In this study, they determined the structure of nucleosomes of a species thought to be endemic to Icelandic hot springs.
“One surprising thing we have learned from our studies of archaeal chromatin is that the way DNA is bent into shape by histones is older than eukaryotes,” Luger says. “This principle is being used by an ancient domain of life, the archaea. But unlike in eukaryotes, were we have defined particles, archaea have kind of 'continuous nucleosomes' that look like a slinky. What really surprised me was how similar the organizing principle is between these two domains of life that are separated by billions of years.” The findings were reported in Science in 2017.
Above: In archaea, the histone-DNA complex can vary in the length of wrapped DNA, as shown here, while eukaryotic nucleosomes have about 2 DNA wraps each. (Images courtesy of K.Luger and CU Boulder)
Other projects include looking at how a dividing cell makes and assembles new nucleosomes for the newly replicated genome. The Luger lab is also interested in DNA repair proteins targeted by promising new anticancer drugs, seeking to learn how proteins know the DNA is damaged and then how the proteins help repair the damage. One project aims to develop an “outlandish tool” to target nucleosomes for gene editing. “It’s always fun to have at least one ‘crazy’ project,” she says. When taking on new lab members, Luger looks for new perspectives, such as enzymologists, “hard core physicists,” or cell biologists, who look at things differently. It’s one way of fighting what she considers the most dangerous threat to science in any lab: Confirmation bias, or the innate human tendency to interpret results in a way that confirms one's hypotheses, especially when results contradict each other are unexpectedly confusing. “I find myself all the time saying let’s assume the whole premise is wrong and let’s rearrange our assumptions,” Luger says. “I don’t believe results lie. It’s literally like a puzzle. A couple of pieces kind of look like they should fit but they don’t, and we have to start over again.” “That’s my mantra,” Luger says. “Problems are three-dimensional. You have to walk around and look at them from another viewpoint. You have to constantly check your premise. Even if nothing fits, people (including me) are reluctant to let it go. I keep telling my coworkers: Your results are telling you something. You have to listen. If they are confusing, it’s not the results’ fault. I really do believe the systems we’re studying are trying to communicate with us: ‘I’m right here. This is what I’m doing.’ I feel their desperation, which is mutual. Most people in lab are quite sick of hearing me say this, I am quite certain.”
-Carol Cruzan Morton
