Wednesday, October 29, 2014
Center for Student Services 360 (Workshop Space) – Center for Student Services

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Figuring Out How We Get the Nitrogen We Need

Caltech Chemists Image Nitrogenase's Active Site At Work

Nitrogen is an essential component of all living systems, playing important roles in everything from proteins and nucleic acids to vitamins. It is the most abundant element in Earth's atmosphere and is literally all around us, but in its gaseous state, N2,, it is inert and useless to most organisms. Something has to convert, or "fix," that nitrogen into a metabolically usable form, such as ammonia. Until about 100 years ago when an industrial-scale technique called the Haber-Bosch process was developed, bacteria were almost wholly responsible for all nitrogen fixation on Earth (lightning and volcanoes fix a small amount of nitrogen). Bacteria accomplish this important chemical conversion using an enzyme called nitrogenase.

"For decades, we have been trying to understand how nitrogenase can interact with this inert gas and carry out this transformation," says Doug Rees, Caltech's Roscoe Gilkey Dickinson Professor of Chemistry and an investigator with the Howard Hughes Medical Institute (HHMI). To fix nitrogen in the laboratory, the Haber-Bosch process requires extremely high temperatures and pressures, yet bacteria are able to complete the conversion under physiological conditions. "We'd love to understand how they do this," he says. "It's a great chemical mystery."

But cracking that mystery has proven extremely difficult using standard chemical techniques. We know that the enzyme is made up of two proteins, the molybdenum iron (MoFe-) protein and the iron (Fe-) protein, which are both required for nitrogen fixation. We also know that the MoFe-protein consists of two metal centers and that one of those is the FeMo-cofactor (also known as  "the cofactor") at the active site, where the nitrogen binds and the chemical transformation takes place.

In 1992, Rees and his graduate student, Jongsun Kim (PhD '93), were the first to determine the structure of the MoFe-protein using X-ray crystallography.

"I think that there was a feeling that once you solved the structure, you'd understand how it worked," Rees says. "What we can say 22 years later is that was certainly not the case."

The dream would be to have atmospheric nitrogen bind to the FeMo-cofactor and to stop time so that chemists could sneak a peak at the chemical structure of the protein at that intermediate point. Since it is not possible to freeze time and because the reaction proceeds too quickly to study by standard crystallographic methods, researchers have come up with an alternative. Chemists have been trying to get carbon monoxide, an inhibitor that halts the enzyme's activity but also closely mimics the structure and electronic makeup of N2, to bind to the cofactor and to then crystallize the product relatively quickly so that the structure can be analyzed using X-ray crystallography.

Unfortunately, the cofactor has stubbornly refused to cooperate. "We've demonstrated more times than we'd like that the form of this protein as isolated doesn't bind substrates," explains Rees. "Usually if you want to know how something binds to a protein, you just add it to your protein and study the crystal structure with X-ray crystallography. But we just couldn't get anything bound to this cofactor."

But in order for the cofactor to exist in a form that would bind to a substrate or an inhibitor, several other conditions must be met—for example, the Fe-protein has to be there. In addition, ATP—a molecule that provides energy for many life processes—must be present, along with yet another enzyme system that regenerates the ATP consumed in the reaction and a source of electrons. So although the aim in crystallography is typically to isolate a purified protein, the chemists had to muddy their samples by adding all these other needed components.

After joining Rees's group as a postdoctoral scholar in 2012, Thomas Spatzal spent months working on this problem, tweaking the method he used for trying to get the carbon monoxide to bind to the cofactor and for crystallizing the product. He adjusted parameters such as the protein concentrations, the temperature under which the samples were prepared, and the amount of time he allowed for the crystals to form. Every week, he sent a new set of crystals, frozen with liquid nitrogen, to be analyzed on an X-ray beamline at the Stanford Synchrotron Radiation Lightsource (SSRL) constructed as part of Caltech's Molecular Observatory with support from the Gordon and Betty Moore Foundation. And every week he worked up the data that came back and looked to see if any of the carbon monoxide bound to the active site.

"People have been seeing the resting state of the active site, where nothing was bound, for years," Spatzal says. "It's always the same thing. It never looks any different."

But on a recent Friday morning, Spatzal processed the latest batch of data, and lo and behold, he finally saw what he had been looking for.

"There was a moment where I looked at it and said, 'Hold on. Something looks different there,'" says Spatzal. "I wondered, 'Am I crazy?' You just don't expect it at first."

What he saw was a first—a crystal structure revealing carbon monoxide bound to the FeMo-cofactor. Spatzal, Rees, and their colleagues describe that structure and their methodology in the September 26 issue of the journal Science.

Spatzal figured out a way to optimize the crystallization process by using tiny crystal seeds to accelerate the rate of crystal growth and conducting all manipulations in the presence of carbon monoxide, allowing him to grow nice crystals of the MoFe-protein and then to see where the carbon monoxide was bound to the cofactor.

What he found was surprising. The carbon monoxide took the place of one of the sulfur atoms in the cofactor's original structure, bridging two of its iron atoms. Many people had expected that the carbon monoxide would bind differently, so that it would stick out, adding extra density to the structure. But because it displaced the sulfur, the cofactor only took on a slightly different arrangement of atoms.

In addition, Spatzal showed that when the carbon monoxide is removed, the sulfur can reattach, reactivating the cofactor so that it can once again fix nitrogen.

"As astonishing as this structure was—that the carbon monoxide replaced the sulfur—I think it's even more astonishing that Thomas was able to establish that the cofactor could be reactivated," Rees says. "I don't think anyone had imagined that you would get this sort of rearrangement of the cofactor as part of the interaction."

"You could imagine that if you put an inhibitor on a system, it could damage the metal center and inactivate the protein so that it would no longer do its job. The fact that we can get it back into an active state means that it's not permanently damaged, and that has physiological meaning in terms of how nitrogen fixation occurs in nature," says Spatzal.

The researchers note that this result would still be a long way off without the X-ray crystallography resources of Caltech's Molecular Observatory, which has abundant dedicated time on a beamline at SSRL. "We were really fortunate that the Moore Foundation funded this access to the beamline," says Rees. "That was really essential for this project because it took a lot of optimization to work everything out. We were able to keep regularly sending samples and right away get feedback about how things were working. It's an unbelievable resource."

Additional Caltech authors on the paper, "Ligand binding to the FeMo-cofactor: Structures of CO-bound and reactivated nitrogenase," are Kathryn A. Perez, a graduate student, and James Howard, a visiting associate who is also affiliated with the University of Minnesota and where Rees was a postdoc. Oliver Einsle of the Institut fur Biochemie in Freiburg, Germany, and the Albert-Ludwigs-Universität Freiburg, was a postdoc with Rees as well as Spatzal's thesis advisor and is a coauthor on the paper. Spatzal is an associate with HHMI.

This work was supported by grants from the National Institutes of Health, Deutsche Forschungsgemeinschaft, and the European Research Council N-ABLE project. The Molecular Observatory is supported by the Gordon and Betty Moore Foundation, the Beckman Institute, and the Sanofi-Aventis Bioengineering Research Program at Caltech. Microbiology research at Caltech is supported by the Center for Environmental Microbial Interactions

Kimm Fesenmaier
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Stoltz Wins Synthetic Chemistry Award

Professor of Chemistry Brian Stoltz has been named the winner of the 2015 Mukaiyama Award from the Society of Synthetic Organic Chemistry, Japan. He was noted for "the discovery and development of new reactions and processes for the synthesis of natural products and non-naturally occurring bioactive structures," according to the award citation.

Stoltz's work is aimed at developing new strategies for creating complex molecules with interesting structural, biological, and physical properties. The goal is to use these complex molecules to guide the development of new reaction methodology to extend fundamental knowledge and to potentially lead to useful biological and medical applications.

The Mukaiyama Award, named in honor of Japanese synthetic chemist Teruaki Mukaiyama, comes with a $5,000 award, a medallion, and a certificate. It is given to an individual 45 years old or younger who has made outstanding contributions to synthetic organic chemistry. Each year, two winners are selected, one each from Japanese and non-Japanese nominees. Also being honored this year is Professor Shigehiro Yamaguchi of Nagoya University.


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In Our Community
Wednesday, October 29, 2014
Avery Courtyard – Avery House

Fall Family Festival

Friday, October 17, 2014
Center for Student Services 360 (Workshop Space) – Center for Student Services

TA Training: fall make-up session

Alumnus Eric Betzig Wins 2014 Nobel Prize in Chemistry

Eric Betzig (BS '83), a group leader at the Howard Hughes Medical Institute's Janelia Farm Research Campus in Ashburn, Virginia, has been awarded the 2014 Nobel Prize in Chemistry along with Stefan W. Hell of the Max Planck Institute for Biophysical Chemistry and William E. Moerner of Stanford University. The three were honored "for the development of super-resolved fluorescence microscopy," a method that allows for the creation of "super-images" with a resolution on the order of nanometers, or billionths of a meter. In essence, the work turns microscopy into "nanoscopy."

The technique developed by the trio overcomes the so-called Abbe diffraction limit, which describes a physical restriction on the sizes of the structures that can be resolved using optical microscopy, showing that, essentially, nothing smaller than one-half the wavelength of light, or about 0.2 microns, can be discerned by these scopes. The result of the Abbe limit is that only the larger structures within cells—organelles like mitochondria, for example—can be resolved and studied with regular microscopes but not individual proteins or even viruses. The restriction is akin to being able to observe the buildings that make up a city but not the city's inhabitants and their activities.

Betzig, building on earlier work by Hell and Moerner, found that it was possible to work around the Abbe limit to create very-high-resolution images of a sample, such as a developing embryo, by using fluorescent proteins that glow when illuminated with a weak pulse of light. Each time the sample is illuminated, a different, sparsely distributed subpopulation of fluorescent proteins will light up and, because the glowing molecules are spaced farther apart than the Abbe diffraction limit, a standard microscope would be able to capture them. Still, each of the images produced in this way has relatively low resolution—that is, they are blurry. Betzig, however realized that by superimposing many such images, he would be able to obtain a sharp super-image, in which nanoscale structures are clearly visible. The new technique was first described in a 2006 paper published in the journal Science.

After Caltech, Betzig, a physics major from Ruddock House, earned an MS (1985) and a PhD (1988) from Cornell University. He worked at AT&T Bell Laboratories until 1994, when he stepped away from academia and science to work for his father's machine tool company. Betzig returned to research in 2002 and joined Janelia in 2005.

To date, 33 Caltech alumni and faculty have won a total of 34 Nobel Prizes. Last year, alumnus Martin Karplus (PhD '54) also received the Chemistry Prize. 

Kathy Svitil
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In Our Community

Orchestrating the Healing Process in a Damaged Cornea

It is safe to say that the eye is an amazing biological system. One reason is its keratocyte cells—specialized cells that make up the bulk of the cornea. Unlike most of the other cells in our body, those in the cornea are transparent, making sight possible. Should something happen to make the cornea opaque, blindness results.

Sadly, injuries to the cornea do occur, sometimes in the simplest of ways, such as getting sand in one's eyes and scratching the cornea. The scar tissue that then grows to heal the cornea may have the unwanted side effect of being opaque. This does not happen, however, if the cornea and the tissue around it heal in a very orderly fashion. The question, then: Is it possible to encourage this orderly healing after an injury, thus preserving vision?

Professor of Chemical Engineering Julia Kornfield and graduate student Amy Fu are very much hoping that this is the case. To find out, they have assigned a few students, including Caltech senior and recent SURF fellow Jacqueline Masehi-Lano, to experiment with various growth factors that might inhibit the formation of scar tissue and promote orderly wound healing.

"We chose three growth factors to test because Amy Fu and I read several papers on growth factors that have been able to suppress some types of scar tissue," Masehi-Lano says. "In particular, we want to inhibit the formation of alpha smooth muscle actin, the type of stress fiber that creates opaque scars over corneal wounds. So far, the experiments I've done with cell cultures have worked pretty well, so it looks promising."

Eventually, the researchers hope to encapsulate the growth factors in a hydrogel that is reminiscent of the native cornea. "Our hydrogel starts out as a liquid and gels in situ on the eye," explains Masehi-Lano.

Masehi-Lano is enthusiastic about her experience with the SURF program. This past summer was her second in Kornfield's lab, and last year she was a recipient of an Amgen scholarship. "I'm really grateful that my mentor and my co-mentor have entrusted me with my own project and have allowed me to conduct my own experiments. And since it was my second summer in this lab, I was able to take up a leadership role by training a new SURF student," she says. "For me, SURF has gone beyond research. I've been able to improve my ability to present my research to the general public, which I think is extremely important." Indeed, Masehi-Lano was awarded the Caltech Doris S. Perpall SURF Speaking Competition for delivering the most outstanding oral research presentation.

Masehi-Lano plans to continue in bioengineering and is contemplating an MD/PhD program. "I've always been interested in the medical field, and though I'm committed to doing research," she explains, "I'd like to be able to do clinical trials and directly apply new medical technologies to people."

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Tuesday, October 7, 2014
Red Door Cafe – Winnett Student Center

Samba and Salsa Exhibition

Caltech Researchers Receive NIH BRAIN Funding

On September 30, the National Institutes of Health (NIH) announced its first round of funding in furtherance of President Obama's "Brain Research through Advancing Innovative Neurotechnology"—or BRAIN—Initiative. Included among the 58 funded projects—all of which, according to the NIH, are geared toward the development of "new tools and technologies to understand neural circuit function and capture a dynamic view of the brain in action"—are six projects either led or co-led by Caltech researchers.

The Caltech projects are:

"Dissecting human brain circuits in vivo using ultrasonic neuromodulation"

Doris Tsao, assistant professor of biology
Mikhail Shapiro, assistant professor of chemical engineering

Tsao and Shapiro are teaming up to develop a new technology that both uses ultrasound to map and determine the function of interconnected brain networks and, ultimately, to change neural activity deep within the brain. "This would open new horizons for understanding human brain function and connectivity, and create completely new options for the noninvasive treatment of brain diseases such as intractable epilepsy, depression, and Parkinson's disease," Tsao says. "The key," Shapiro adds, "is to gain a precise understanding of the various mechanisms by which sound waves interact with neurons in the brain so we can use ultrasound to produce very specific neurological effects. We will be able to do this across the full spectrum, from molecules up to large model organisms."

"Modular nanophotonic probes for dense neural recording at single-cell resolution"

Michael Roukes, Robert M. Abbey Professor of Physics, Applied Physics, and Bioengineering
Thanos Siapas, professor of computation and neural systems

Roukes, Siapas, and their colleagues at Columbia University and Baylor College of Medicine propose to build ultra-dense arrays of miniature light-emitting and light-sensing probes using advanced silicon "chip" technology that permits their production en masse. These probes open the new field of integrated neurophotonics, Roukes says, and will permit simultaneous recording of the electrical activity of hundreds of thousands to, ultimately, millions of neurons, with single-cell resolution, in any given region of the brain. "The instrumentation we'll develop will enable us to observe the trafficking of information, in vivo, in brain circuits on an unprecedented scale, and to correlate this activity with behavior," he says.

"Time-Reversal Optical Focusing for Noninvasive Optogenetics"

Changhuei Yang, professor of electrical engineering, bioengineering, and medical engineering
Viviana Gradinaru, assistant professor of biology

Deep-brain stimulation has been used successfully for nearly two decades for the treatment of epilepsy, Parkinson's disease, chronic pain, depression, and other disorders. Current systems rely on electrodes implanted deep within the brain to modify the firing pattern of specific clusters of neurons, bringing them back into a more normal pattern. Yang and Gradinaru are working together on a method that would use only light waves to noninvasively activate light-sensitive molecules and precisely guide the firing of nerves. Biological tissues are opaque due to the scattering of light waves, and that scattering makes it impossible to finely focus a laser beam deep into brain tissue. The researchers hope to use an optical "time-reversal" trick previously developed by Yang to counteract the scattering, allowing light beams to be targeted to specific locations within the brain. "The technology to be developed in this project has the potential for wide-ranging applications, including noninvasive deep brain stimulation and precise incisionless laser surgery," he says.

"Integrative Functional Mapping of Sensory-Motor Pathways"

Michael H. Dickinson, Esther M. and Abe M. Zarem Professor of Bioengineering

As in other animals, locomotion in the fruit fly is a complicated process involving the interplay of sensory systems and motor circuits in the brain. Dickinson and his colleagues hope to decipher just how the brain uses sensory information to guide movements by developing a system to record the activity of large numbers of individual neurons from across the brains of fruit flies, as the flies fly in flight simulator or walk on a treadmill and are simultaneously exposed to various sights and sounds. Understanding sensory–motor integration, he says, should lead to a better understanding of human disorders, including Parkinson's disease, stroke, and spinal cord injury, and aid in the design and optimization of robotic prosthetic limbs and prosthetic devices that restore sight and other senses.

"Establishing a Comprehensive and Standardized Cell Type Characterization Platform"

David J. Anderson, Seymour Benzer Professor of Biology; Investigator, Howard Hughes Medical Institute (co-PI)

In collaboration with Hongkui Zeng and colleagues at the Allen Institute for Brain Science in Seattle, Anderson will help to develop a detailed, publicly available database characterizing the genetic, physiological, and morphological features of the various cell types in the brain that are involved in circuits controlling sensations and emotions. Understanding the cellular building blocks of brain circuits, the researchers say, is crucial for figuring out how those circuits can malfunction in disease. In particular, Anderson's lab will focus on the cells of the brain's hypothalamus and amygdala—structures that are vital to emotions and behavior, and involved in human psychiatric disorders such as post-traumatic stress disorder, anxiety, and depression. "This project will serve as a model for hub-and-spoke collaborations between academic laboratories and the Allen Institute, permitting access to their valuable resources and technologies while advancing the field more broadly," Anderson says.

"Vertically integrated approach to visual neuroscience: microcircuits to behavior"

Markus Meister, Lawrence A. Hanson, Jr. Professor of Biology (co-PI)

This project, led by Hyunjune Sebastian Seung of Princeton University, will use genetic, electrophysiological, and imaging tools to identify and map the neural connections of the retina, the light-sensing tissue in the eye, and determine their roles in visual perception and behavior. "Here we are shooting for a vertically integrated understanding of a neural system," Meister says. "The retina offers such a fantastic degree of experimental access that one can hope to bridge all scales of organization, from molecules to cells to microcircuits to behavior. We hope that success here can eventually serve as a blueprint for understanding other parts of the brain." Knowing the neural mechanisms for vision can also influence technological applications, such as new algorithms for computer vision, or the development of retinal prostheses for the treatment of blindness.

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In Our Community
Tuesday, October 7, 2014
Center for Student Services 360 (Workshop Space) – Center for Student Services

Thirty Meter Telescope Groundbreaking and Blessing


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