From Rosetta@home via Bloomberg View: “Protein Engineering May Be the Future of Science”

Rosetta@home

Bloomberg View

March 27, 2018
Faye Flam

Some scientists think designing new proteins could become as significant as tweaking DNA.

Let’s build a better sperm whale. Photograph: SSPL/Getty Images

Scientists are increasingly betting their time and effort that the way to control the world is through proteins. Proteins are what makes life animated. They take information encoded in DNA and turn it into intricate three-dimensional structures, many of which act as tiny machines. Proteins work to ferry oxygen through the bloodstream, extract energy from food, fire neurons, and attack invaders. One can think of DNA as working in the service of the proteins, carrying the information on how, when and in what quantities to make them.

Living things make thousands of different proteins, but soon there could be many more, as scientists are starting to learn to design new ones from scratch with specific purposes in mind. Some are looking to design new proteins for drugs and vaccines, while others are seeking cleaner catalysts for the chemical industry and new materials.

David Baker, director for the Institute for Protein Design at the University of Washington, compares protein design to the advent of custom tool-making. At some point, proto-humans went beyond merely finding uses for pieces of wood, rock or bone, and started designing tools to suit specific needs — from screwdrivers to sports cars.

See the full article here.

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Rosetta@home needs your help to determine the 3-dimensional shapes of proteins in research that may ultimately lead to finding cures for some major human diseases. By running the Rosetta program on your computer while you don’t need it you will help us speed up and extend our research in ways we couldn’t possibly attempt without your help. You will also be helping our efforts at designing new proteins to fight diseases such as HIV, Malaria, Cancer, and Alzheimer’s (See our Disease Related Research for more information). Please join us in our efforts! Rosetta@home is not for profit.

About Rosetta

One of the major goals of Rosetta is to predict the shapes that proteins fold up into in nature. Proteins are linear polymer molecules made up of amino acid monomers and are often refered to as “chains.” Amino acids can be considered as the “links” in a protein “chain”. Here is a simple analogy. When considering a metal chain, it can have many different shapes depending on the forces exerted upon it. For example, if you pull its ends, the chain will extend to a straight line and if you drop it on the floor, it will take on a unique shape. Unlike metal chains that are made of identical links, proteins are made of 20 different amino acids that each have their own unique properties (different shapes, and attractive and repulsive forces, for example), and in combination, the amino acids exert forces on the chain to make it take on a specific shape, which we call a “fold.” The order in which the amino acids are linked determines the protein’s fold. There are many kinds of proteins that vary in the number and order of their amino acids.

To predict the shape that a particular protein adopts in nature, what we are really trying to do is find the fold with the lowest energy. The energy is determined by a number of factors. For example, some amino acids are attracted to each other so when they are close in space, their interaction provides a favorable contribution to the energy. Rosetta’s strategy for finding low energy shapes looks like this:

Start with a fully unfolded chain (like a metal chain with its ends pulled).
Move a part of the chain to create a new shape.
Calculate the energy of the new shape.
Accept or reject the move depending on the change in energy.
Repeat 2 through 4 until every part of the chain has been moved a lot of times.

We call this a trajectory. The end result of a trajectory is a predicted structure. Rosetta keeps track of the lowest energy shape found in each trajectory. Each trajectory is unique, because the attempted moves are determined by a random number. They do not always find the same low energy shape because there are so many possibilities.

A trajectory may consist of two stages. The first stage uses a simplified representation of amino acids which allows us to try many different possible shapes rapidly. This stage is regarded as a low resolution search and on the screen saver you will see the protein chain jumping around a lot. In the second stage, Rosetta uses a full representation of amino acids. This stage is refered to as “relaxation.” Instead of moving around a lot, the protein tries smaller changes in an attempt to move the amino acids to their correct arrangment. This stage is regarded as a high resolution search and on the screen saver, you will see the protein chain jiggle around a little. Rosetta can do the first stage in a few minutes on a modern computer. The second stage takes longer because of the increased complexity when considering the full representation (all atoms) of amino acids.

Your computer typically generates 5-20 of these trajectories (per work unit) and then sends us back the lowest energy shape seen in each one. We then look at all of the low energy shapes, generated by all of your computers, to find the very lowest ones. This becomes our prediction for the fold of that protein.

To join this project, download and install the BOINC software on which it runs. Then attach to the project. While you are at BOINC, look at some of the other projects to see what else might be of interest to you.

U Washington Dr. David Baker

My BOINC

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From Rutgers: “Rutgers Scientists Discover ‘Legos of Life'”

Rutgers University

THIS POST IS DEDICATED TO J.L. FROM HP AND RUTGERS. I HOPE HE SEES IT.

January 21, 2018
Todd B. Bates

Deep dive into the 3D structures of proteins reveals key building blocks.

Rutgers researchers identified a small set of simple protein building blocks (left) that likely existed at the earliest stages of life’s history. Over billions of years, these “Legos of life” were assembled and repurposed by evolution into complex proteins (right) that are at the core of modern metabolism.
Image: Vikas Nanda/Rutgers Robert Wood Johnson Medical School.

Rutgers scientists have found the “Legos of life” – four core chemical structures that can be stacked together to build the myriad proteins inside every organism – after smashing and dissecting nearly 10,000 proteins to understand their component parts.

The four building blocks make energy available for humans and all other living organisms, according to a study published online today in the Proceedings of the National Academy of Sciences.

The study’s findings could lead to applications of these stackable, organic building blocks for biomedical engineering and therapeutic proteins and the development of safer, more efficient industrial and energy catalysts – proteins and enzymes that, like tireless robots, can repeatedly carry out chemical reactions and transfer energy to perform tasks.

“Understanding these parts and how they are connected to each other within the existing proteins could help us understand how to design new catalysts that could potentially split water, fix nitrogen or do other things that are really important for society,” said Paul G. Falkowski, study co-author and a distinguished professor who leads the Environmental Biophysics and Molecular Ecology Laboratory at Rutgers University–New Brunswick.

The scientists’ research was done on computers, using data on the 3D atomic structures of 9,500 proteins in the RCSB Protein Data Bank based at Rutgers, a rich source of information about how proteins work and evolve.

“We don’t have a fossil record of what proteins looked like 4 billion years ago, so we have to take what we have today and start walking backwards, trying to imagine what these proteins looked like,” said Vikas Nanda, senior author of the study and an associate professor in the Department of Biochemistry and Molecular Biology at Rutgers’ Robert Wood Johnson Medical School, within Rutgers Biomedical and Health Sciences. “The study is the first time we’ve been able to take something with thousands of amino acids and break it down into reasonable chunks that could have had primordial origins.”

The identification of four fundamental building blocks for all proteins is just a beginning. Nanda said future research may discover five or 10 more building blocks that serve as biological Legos.

“Now we need to understand how to put these parts together to make more interesting functional molecules,” he said. “That’s the next grand challenge.”

The study’s lead author is Hagai Raanana, a post-doctoral associate in the Environmental Biophysics and Molecular Ecology Program. Co-authors include Douglas H. Pike, a doctoral student at the Rutgers Institute for Quantitative Biomedicine, and Eli K. Moore, a post-doctoral associate in the Environmental Biophysics and Molecular Ecology Program.

See the full article here .

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From UCLA Newsroom: “Imaging technique could be ‘new ballgame’ in drug development”

UCLA Newsroom

January 02, 2018
Tami Dennis

UCLA researcher Tamir Gonen explores the potential of MicroED in neurological diseases.

Courtesy of Tamir Gonen
Tamir Gonen published his proof of principle paper [eLIFE] on MicroED in 2013.

Biochemistry and structural biology are surprisingly — at least to the uninitiated — visual fields. This is especially true in the study of proteins. Scientists like to see the structure of proteins within cells to help them truly understand how they work, how they don’t work or how they can be modified to work as they should. That is, how they can be targeted with drugs to cure disease.

Current methods, however, have their downsides. Many widely used techniques require large amounts of protein for analysis, even though many diseases are caused by proteins that are far from abundant or that are difficult to amass in large quantities. A new method pioneered by a professor who recently joined UCLA overcomes this challenge, offering untold potential in the exploration of disease and treatment.

Called “MicroED,” for micro-electron diffraction, the technique uses high-powered electron microscopy to determine the structure of proteins with atomic precision, using samples that are only one-billionth the size required by other imaging methods.

Tamir Gonen, a new professor of physiology and biological chemistry at the David Geffen School of Medicine at UCLA, is the developer of MicroED. For the past five years, Gonen has been spearheading the exploration of MicroED in his lab at the Janelia Research Campus of the Howard Hughes Medical Institute near Washington D.C.

Now, in joining UCLA’s faculty, Gonen’s goal is to set up a lab centered on this new imaging tool. Already the university is using the technique in the labs of Jose Rodriguez, assistant professor of biochemistry, and David Eisenberg, a professor of chemistry and biochemistry and of biological chemistry. Both use MicroED to view the structures of proteins involved in neurodegeneration.

“With MicroED, the way we think about disease is going to be different,” Gonen said. “Because it uses minute samples and the resolution we get is very high, problems that were beyond our reach are suddenly attainable. We can see individual atoms and even peer deeper into subatomic space and see things that have not been seen before.”

Samples used in MicroED resemble jewels with one exception, they are made out of biological material rather than precious mineral. Gonen lab.

Gonen began working on the technique at the Howard Hughes Medical Institute Janelia Research Campus, where he was a group leader. Upon moving to UCLA Gonen is now an investigator of the Howard Hughes Medical Institute and professor of physiology and biological chemistry. Prior to that, he was an assistant professor, then a tenured associate professor, at the University of Washington School of Medicine, as well as a Howard Hughes Medical Institute Early Career Scientist.

Since 2013, which marked his publication of a proof of principle paper on MicroED [eLIFE], Gonen has been an advocate for the technique. As other researchers have come to understand the long-sought imaging potential MicroED offers, about 20 institutions worldwide have begun setting up MicroED labs, many with Gonen’s help.

“I have fantastic folks working in my lab, and they are extremely collegial and want to help others get their science done,” Gonen said.

That focus on getting “science done” — and MicroED itself — has enormous ramifications for the treatment of HIV, Parkinson’s, Alzheimer’s and other neurodegenerative diseases. “When you’re talking about drug discovery, it’s a whole new ballgame,” Gonen said.

Gonen’s development of MicroED stemmed from his study of cell membranes, specifically the protein gateways within those membranes.

These gateways can help cells maintain healthy homeostasis in which everything works as it should — think of it as a biological “peace.” When things go wrong, however, as happens with disease, these gateways might allow too much of one substance, such as water or sugar, in or out.

This illustration of a protein shows an example of a structure that could only be determined by the capabilities of micro-electron diffraction. Gonen lab.

Gonen knew that targeting these gateways — or targeting their function — could lead to innovative ways to control disease and help patients. But he and other scientists needed good images to facilitate the design of better drugs.

“More than 90 percent of all medicines sold these days target G-protein coupled receptors,” Gonen said. “When you feel pain, when you see light, when you taste, when you have any neurological sensation, all of this is occurring because of these receptors.”

Another potential, and timely, target of this type: opioid receptors. “Opioids are an increasingly challenging problem, and not surprisingly there exists an opioid receptor,” Gonen said.

MicroED makes it possible to image these G-protein coupled receptors, which move around a lot. This movement has made it difficult for traditional methods to capture images of them.

MicroED’s potential goes far beyond such receptors, however.

Larry Zipursky, a distinguished professor biological chemistry and the leader of the neuroscience research theme at the David Geffen School of Medicine at UCLA, called the approach “revolutionary.”

“This technique uses a rational approach to disease,” Zipursky said. “It allows researchers to assess the structure of abnormal proteins that give rise to disease; from this structural determination, they can assess the disease in a more strategic way.”

Gonen is enthusiastic about the larger potential as well. “For a medical school, this is going to be quite a resource for pushing research forward. I’m hoping to collaborate with a lot of people.”

In fact, he’s already working on several projects, including one that points to a way to make more efficient HIV medicines. This is in addition to the projects with Eisenberg and Rodriguez, who’s studying the conversion of proteins from a normal state into an abnormal, clumped — or aggregated — state, as seen in Alzheimer’s and other diseases of the brain and nervous system.

“We need new and better treatments for neurodegenerative diseases, one way to achieve this is to understand how atomic scale changes in the brain lead to disease. What do the structures look like before and after, and, quite simply, how are they so toxic?” Rodriguez said. “MicroED may finally open the door to that understanding.”

The potential of such collaborations is what brought Gonen — and his emphasis on MicroED — to UCLA.

“What I like about UCLA is it’s a top-rate institution — you can find some expert in every field,” Gonen said. “They’re a world leader, but they come without the ego you may find at other institutions.”

That collaboration, he’s convinced, will lead to new approaches, new discoveries and new cures.

Further papers:
>Structure of catalase determined by MicroED
Taking the measure of MicroED
Protein structure determination by MicroED

See the full article here .

Please help promote STEM in your local schools.

Stem Education Coalition

For nearly 100 years, UCLA has been a pioneer, persevering through impossibility, turning the futile into the attainable.

We doubt the critics, reject the status quo and see opportunity in dissatisfaction. Our campus, faculty and students are driven by optimism. It is not naïve; it is essential. And it has fueled every accomplishment, allowing us to redefine what’s possible, time after time.

This can-do perspective has brought us 12 Nobel Prizes, 12 Rhodes Scholarships, more NCAA titles than any university and more Olympic medals than most nations. Our faculty and alumni helped create the Internet and pioneered reverse osmosis. And more than 100 companies have been created based on technology developed at UCLA.

From Salk: “Revealing the best-kept secrets of proteins”

Salk Institute for Biological Studies

December 14, 2017
No writer credit

From left: John Lubin, Vicki Lundblad and Tim Tucey. Credit: Salk Institute

Salk scientists develop new approach to identify important undiscovered functions of proteins.

In the bustling setting of the cell, proteins encounter each other by the thousands. Despite the hubbub, each one manages to selectively interact with just the right partners, thanks to specific contact regions on its surface that are still far more mysterious than might be expected, given decades of research into protein structure and function.

Now, Salk Institute scientists have developed a new method to discover which surface contacts on proteins are critical for these cellular interactions. The novel approach shows that essential new functions can be uncovered even for well-studied proteins, and has significant implications for therapeutic drug development, which depends heavily on how drugs physically interact with their cellular targets. The paper appeared in the early online version of Genetics in late November, and is slated for publication in the January print edition of the journal.

“This paper illustrates the power of this methodology,” says senior author Vicki Lundblad, holder of the Ralph S. and Becky O’Conner Chair. “It can not only identify previously undiscovered activities for a protein, but it can also pinpoint the exact amino acids on a protein surface that perform these new functions.”

Amino acids are the building blocks of proteins. Their specific linear arrangement determines the identity of a protein, and clusters of them on the protein’s surface serve as contacts, regulating how that protein interacts with other proteins and molecules. Lundblad and her colleagues suspected that, despite decades of work deciphering the mysteries of proteins, the extent of this regulatory landscape on the surface of proteins had remained mostly unexplored. Long ago, her group unexpectedly discovered one such regulatory amino acid cluster, while searching one-by-one through 300,000 mutant yeast cells. Although that work opened up a new area of research in the field of telomere biology, Lundblad was determined to figure out a more robust methodology that could rapidly uncover many more of these unexplored protein surfaces.

Enter John Lubin, now a PhD student in Lundblad’s lab, who began working with her as an undergraduate.

“My task was to figure out how to search through 30 mutant yeast cells, instead of 300,000, to discover new activities for a protein,” says Lubin, the paper’s co–first author. Timothy Tucey, the other co–first author, was a postdoctoral researcher in Lundblad’s group and is now at Monash University.

Together they turned to a protein called Est1, which Lundblad had discovered in yeast as a postdoctoral researcher in 1989. Est1 is a subunit of a protein (an enzyme) called telomerase, which keeps the protective caps at the ends of chromosomes (known as telomeres) from getting too short. As the first subunit of telomerase to be discovered, Est1 has been subjected to intensive study by many research groups.

The Salk team’s approach involved introducing a small, but customized, set of mutations into yeast cells that would selectively disrupt surface contacts on the cells’ Est1 protein. The team then analyzed the cells to see what effect, if any, the various mutations had. Abnormalities resulting from a specific mutation would suggest what the role of the unmutated version was. To do so, they used a genetic trick, by flooding the cells with each mutant protein, and looking for the rare mutant protein that could interfere with cell function, as their previous work had shown that this would preferentially target the protein surface.

Lundblad’s team discovered four functions for Est1 through this approach. Impairment of any of these four functions by mutations to Est1’s surface amino acids, the scientists found, resulted in cells that had critically short telomeres, indicating specific roles for the Est1 contacts in the telomerase complex.

“What has us excited about this technique is that it can be applied to numerous proteins,” says Lundblad. “In particular, many therapeutic drugs rely on being able to access a very specific location on a protein surface, which we suspect can be uncovered by this method.”

Using this approach, her team has already uncovered new functions for a set of proteins that regulate the stability of the genome, and has also applied for grants that fund research into drug targets.

The work was funded by the National Institutes of Health, the National Science Foundation, the Rose Hills Foundation and the Glenn Center for Aging Research.

See the full article here .

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Every cure has a starting point. Like Dr. Jonas Salk when he conquered polio, Salk scientists are dedicated to innovative biological research. Exploring the molecular basis of diseases makes curing them more likely. In an outstanding and unique environment we gather the foremost scientific minds in the world and give them the freedom to work collaboratively and think creatively. For over 50 years this wide-ranging scientific inquiry has yielded life-changing discoveries impacting human health. We are home to Nobel Laureates and members of the National Academy of Sciences who train and mentor the next generation of international scientists. We lead biological research. We prize discovery. Salk is where cures begin.

From IST Austria “Researchers at IST Austria define function of an enigmatic synaptic protein”

IST Austria

November 21, 2017

Synaptotagmin 7 ensures efficiency of inhibitory signal transmission – Study published in Cell Reports

Photo credit: Chen et al, Cell Reports

Communication is often mired in contradiction – also in the brain. Neuroscientists at IST Austria were now able to resolve one such contradiction.

In our brains, neurons communicate by sending chemical signals across their connections, the synapses. The molecular machinery required to send a signal involves not only the signal itself, the neurotransmitter, but a large variety of other proteins that act as sensors, effectors, modulators, and scaffolds. Synaptotagmins are part of this complex machinery. Synaptotagmin proteins come in many flavours: humans and other mammals have 17 different varieties. For most of these proteins, however, scientists do not yet understand the function. A group of neuroscientists led by Peter Jonas, Professor at the Institute of Science and Technology Austria (IST Austria), have now resolved the role of Synaptotagmin 7 during signal transmission at an inhibitory synapse. Writing in Cell Reports, the team, including first author and PhD student Chong Chen as well as researchers at the Max Planck Florida Institute for Neuroscience, shows that Synaptotagmin 7 ensures the efficiency of high-frequency inhibitory synaptic transmission. “The role of Synaptotagmin 7 has been controversial. For the first time, we defined its functional contribution at an inhibitory GABAergic synapse”, explains lead author Peter Jonas.

Conflicting role in signal transmission

Only in January of this year, Jonas and Chen showed that Synaptotagmin 2 is the calcium sensor that makes certain synapses fast – those that use the transmitter GABA. In the current study, the researchers turned their attention to another member of the Synaptotagmin family, Synaptotagmin 7. The brain contains a large amount of Synaptotagmin 7, but so far, scientists were not able to pinpoint the protein’s function. Partly, the reason is a contradiction between the function Synaptotagmin 7 seemed to have, and the characteristics of signal transmission observed.

Synaptotagmin 7 appears to function as a calcium sensor that mediates the release of a variable barrage of neurotransmitter into the synaptic cleft, a phenomenon called asynchronous transmitter release. There are also indications that Synaptotagmin 7 plays a role in facilitation, an increase in neurotransmission at the synapse. But on the other hand Synaptotagmin 7 is also found in large amounts in a class of neurons called fast-spiking, parvalbumin-expressing GABAergic interneurons. These neurons form synapses which appear to contradict the suggested functions of Synaptotagmin 7: the synapses release neurotransmitter in a tightly synchronized manner, rather than asynchronously, and show a reduction in neurotransmission, rather than facilitation during repetitive stimulation. In their study, Chen et al. now resolve this apparent contradiction.

Synaptotagmin 7 regulates information flow in cerebellum

The researchers investigated how signal transmission changes when they delete Synaptotagmin 7 in an inhibitory synapse, the GABAergic synapse between the basket cells (BCs) and Purkinje cells (PCs) in the cerebellum (a brain region required for motor control). They show that, indeed, the function of Synaptotagmin 7 is to contribute to asynchronous transmitter release, replenishment of vesicles containing neurotransmitter, and facilitation. But rather than being mutually exclusive, these three functions appear to coexist at BC-PC synapses. Elucidating the effects of Synaptotagmin 7 required careful analysis, because asynchronous release is small and facilitation is overlaid by depression. However the authors demonstrate a substantial difference in the extent of depression. Little depression occurred in the presence of Synaptotagmin 7, but a lot of depression was found in the absence of Synaptotagmin 7. Thus, Synaptotagmin 7 ensures the efficient and frequency-independent signal transmission at the BC-PC synapse, one of its fundamental properties.

When the researchers looked at the level of neuronal networks, they found that Synaptotagmin 7 allows single basket cells to control the activity of a Purkinje cell. These neurons are the only ones that send information out of the cerebellum. Synaptotagmin 7 therefore has a strategic position to regulate the flow of information in this motor circuit. The researchers also found that Synaptotagmin 7 plays a similar (albeit quantitatively smaller) role at BC synapses in the hippocampus, a seahorse-shaped brain region involved in memory and spatial coding. Peter Jonas summarizes: “We have identified a critical role for Synaptotagmin 7 in maintaining the efficacy of transmission at GABAergic synapses in the cerebellum and hippocampus.”

See the full article here.

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The Institute of Science and Technology Austria (IST Austria) is a young international institute dedicated to basic research and graduate education in the natural and mathematical sciences, located in Klosterneuburg on the outskirts of Vienna. Established jointly by the federal government of Austria and the provincial government of Lower Austria, the Institute was inaugurated in 2009 and will grow to about 90 research groups by 2026.

The governance and management structures of IST Austria guarantee its independence and freedom from political and commercial influences. The Institute is headed by the President, who is appointed by the Board of Trustees and advised by the Scientific Board. The first President of IST Austria is Thomas A. Henzinger, a leading computer scientist and former professor of the University of California at Berkeley and the EPFL Lausanne in Switzerland.

From UCLA Newsroom: “UCLA bioengineers discover mechanism that regulates cells’ ‘powerhouses’”

UCLA Newsroom

November 27, 2017
Matthew Chin

In this artist’s rendering, Dnm1 proteins surrounding a mitochondrion are breaking it up into two. Jaime de Anda/ACS Central Science.

UCLA bioengineers and their colleagues have discovered a new perspective on how cells regulate the sizes of mitochondria, the parts of cells that provide energy, by cutting them into smaller units.

The researchers wrote that this finding, demonstrated with yeast proteins, could eventually be used to help address human diseases associated with an imbalanced regulation of mitochondria size — for example, Alzheimer’s or Parkinson’s diseases. In addition, since having mitochondria that are too small or too large can potentially lead to incurable diseases, it is conceivable that the proteins responsible for this process could be potential targets for future therapies.

The study was published in ACS Central Science and was led by UCLA bioengineering professor Gerard Wong.

Inside the cell, mitochondria resemble the long balloons used to create balloon animals. If the mitochondria are too long, they can get tangled. Their sizes are known to be primarily regulated by two proteins, one of which breaks up longer mitochondria into smaller sizes. They are known as cells’ “powerhouses” as they convert chemical energy from food into a form useful for cells to perform all their functions.

Keeping mitochondria at optimal sizes is important to cells’ health. An insufficient amount of the regulating protein, known as Dnm1, results in the mitochondria getting too long and tangled. Too much Dnm1 results in too many short mitochondria. In both cases, the mitochondria are rendered essentially ineffective as power providers for the cell. This situation could lead to neurodevelopmental disorders or neurodegenerative diseases, such as Alzheimer’s or Parkinson’s.

To better understand this mechanism, the researchers used a machine-learning approach they developed in 2016 to figure out exactly how the proteins break up one mitrochondrion into two smaller ones. They also used a powerful technique called “synchrotron small-angle X-ray scattering” at the Stanford Synchrotron Radiation Lightsource, a U.S. Department of Energy research facility, to see how these proteins deform mitochondrial membranes during this process.

SLAC/SSRL

Before this study, it was thought that these proteins encircled the mitochondria, then cut it in two by simply squeezing tightly. The process, the team discovered, is more subtle.

“When Dnm1 wraps around mitochondria, it has been previously shown that the protein physically tightens and pinches,” said Michelle Lee, a recent UCLA bioengineering doctoral graduate who was advised by Wong and is one of two lead authors of the study. “What we found is that when Dnm1 contacts the mitochondrial surface, it also makes that area of the mitochondrion itself more moldable and easier to undergo cleavage. These two effects work hand in hand to make the process of mitochondrial division efficient.”

The other lead author is Ernest Lee, a graduate student in the UCLA-Caltech Medical Scientist Training Program and a bioengineering graduate student also advised by Wong. He carried out the computational analyses for the experiment.

“Using our machine-learning tool, we were able to discover hidden membrane-remodeling activity in Dnm1, consistent with our X-ray studies,” Lee said. “Interestingly, by analyzing distant relatives of Dnm1, we found that the protein gradually evolved this ability over time.”

“This is a very unexpected result — no one thought these molecules would have a split personality, with both personalities necessary for the biological function,” said Wong, who is also a UCLA professor of chemistry and biochemistry and is a member of the California NanoSystems Institute. “The multifunctional behavior we identified may be the rule rather than the exception for proteins.”

Other authors include Andy Ferguson from the University of Illinois at Urbana-Champaign and Blake Hill from the Medical College of Wisconsin.

The research was supported by the National Science Foundation and the National Institutes of Health, with additional support from the Department of Energy for imaging experiments.

See the full article here .

Please help promote STEM in your local schools.

Stem Education Coalition

For nearly 100 years, UCLA has been a pioneer, persevering through impossibility, turning the futile into the attainable.

We doubt the critics, reject the status quo and see opportunity in dissatisfaction. Our campus, faculty and students are driven by optimism. It is not naïve; it is essential. And it has fueled every accomplishment, allowing us to redefine what’s possible, time after time.

This can-do perspective has brought us 12 Nobel Prizes, 12 Rhodes Scholarships, more NCAA titles than any university and more Olympic medals than most nations. Our faculty and alumni helped create the Internet and pioneered reverse osmosis. And more than 100 companies have been created based on technology developed at UCLA.

From U Washington Medicine: “Mini-protein rapid design opens way to new class of drugs”

University of Washington

September 27, 2017
Leila Gray
206.685.0381
leilag@uw.edu

Scientists at the Institute for Protein Design have created a way to generate thousands of different mini-protein binders as possible drug candidates. The proteins can be custom tailored to specific therapeutic targets. Recently, a set of these proteins were successfully tested in mice against the flu, and another group of these binders was able to protect brain cells against the botulism neurotoxin.

Artist impression of designed mini-protein binders targeting Influenza hemagglutinin to effectively bind and neutralize the virus. Cognition Studio, Daniel-Adriano Silva, Lance Stewart

These computer-designed proteins, which did not previously exist in nature, combine the stability and bioavailability of small molecule drugs with the specificity and potency of larger biologics. They would not require refrigeration, and they likely would be simple for patients to take.

“These mini-protein binders have the potential of becoming a new class of drugs that bridge the gap between small molecule drugs and biologics. Like monoclonal antibodies, they can be designed to bind to targets with high selectivity, but they are more stable and easier to produce and to administer,” said David Baker, who led the multi-institutional research project. He is a professor of biochemistry at the University of Washington School of Medicine and director of the UW Institute for Protein Design.

Dr. David Baker, Baker Lab, U Washington

Baker and his colleagues report their findings in article published online Sept. 27 by the journal Nature.

Aaron Chevalier, Daniel-Adriano Silva and Gabriel J. Rocklin were the lead authors and were all senior fellows at the UW Institute for Protein Design at the time of the project.

The method used a computer platform, called Rosetta, developed by Baker and colleagues at the University of Washington. They designed thousands of short proteins, about 40 amino acids in length, that the Rosetta program predicted would bind tightly to the molecular target.

Rosetta@home project, a project running on BOINC software from UC Berkeley

Rosetta@home BOINC project

BOINC

My BOINC

Because of their small size, these short proteins tend to be extremely stable. They can be stored without refrigeration. They also are more easily administered than large protein drugs, such as monoclonal antibodies.

Previously, such short, protein-binder drugs were typically re-engineered versions of naturally occurring proteins. These, however, tended not to be significantly better than monoclonal antibodies.

Because these mini-proteins binders are original designs, they can be tailored to fit their targets much more tightly and are simpler to modify and refine.

In this study, the researchers sought to design two sets of these proteins: one set that would prevent the influenza virus from invading cells and another that would bind to and neutralize a deadly nerve toxin from botulism. This toxin is considered a potential bioweapon.

The computer modeling identified the amino-acid sequences of thousands of short proteins that would fit into and bind to the influenza and botulinum targets. The researchers created short pieces of DNA that coded each of these proteins, grew the proteins in yeast cells, and then looked at how tightly they bound to their targets. The targets were Influenza H1 hemagglutinin and botulinum neurotoxin B.

All told, the method allowed them to design and test 22,660 proteins in just a few months. More than than two-thousand of them bound to their targets with high affinity.

Evaluation of the best candidates found that the anti-influenza proteins neutralized viruses in cell culture and other designed proteins prevented the botulinum toxin from entering brain cells.

A nasal spray containing one of the custom-designed proteins completely protected mice from the flu if administered before or as much as 72 hours after exposure.. The protection that the treatment provides equaled or surpassed that seen with antibodies, the researchers report.

Testing of a subset of the proteins showed that they were extremely stable and, unlike antibodies, did not become inactivated by high temperatures. The small proteins also triggered little or no immune response, a problem that often renders larger protein drugs ineffective.

Funding for the study came from Life Sciences Discovery Fund Launch grant (9598385), Doctorado en Ciencias Bioquiacutemicas UNAM (R56AI117675), Molecular Basis of Viral Pathogenesis Training Grant (T32AI007354-26A1), Investigator in the Pathogenesis of Infectious Disease award from the Burroughs Wellcome Fund and NIH (1R01NS080833), CoMotion Mary Gates Innovation Fellow; Shenzhen Science and Technology Innovation Committee (JCYJ20170413173837121), Hong Kong Research Grant Council (C6009-15G and AoE/P-705/16), PAPIIT UNAM (IN220516), CONACyT (254514) and Facultad de Medicina UNAM (AI091823, AI123920,AI125704), NIAID grant (1R41AI122431) (1R21AI119258), and Life Sciences Discovery Fund grant (20040757).

See the full article here .

Please help promote STEM in your local schools.

Stem Education Coalition

The University of Washington is one of the world’s preeminent public universities. Our impact on individuals, on our region, and on the world is profound — whether we are launching young people into a boundless future or confronting the grand challenges of our time through undaunted research and scholarship. Ranked number 10 in the world in Shanghai Jiao Tong University rankings and educating more than 54,000 students annually, our students and faculty work together to turn ideas into impact and in the process transform lives and our world. For more about our impact on the world, every day.

So what defines us — the students, faculty and community members at the University of Washington? Above all, it’s our belief in possibility and our unshakable optimism. It’s a connection to others, both near and far. It’s a hunger that pushes us to tackle challenges and pursue progress. It’s the conviction that together we can create a world of good. Join us on the journey.

From Salk: “Protein turnover could be clue to living longer”

Salk Institute for Biological Studies

August 30, 2017

Overactive protein synthesis found in premature aging disease may also play role in normal aging.

Scientists at the Salk Institute found that protein synthesis is overactive in people with progeria. The work, described in Nature Communications on August 30, 2017, adds to a growing body of evidence that reducing protein synthesis can extend lifespan—and thus may offer a useful therapeutic target to counter both premature and normal aging.

“The production of proteins is an extremely energy-intensive process for cells,” says Martin Hetzer, vice president and chief science officer of the Salk Institute and senior author of the paper. “When a cell devotes valuable resources to producing protein, other important functions may be neglected. Our work suggests that one driver of both abnormal and normal aging could be accelerated protein turnover.”

Nucleoli in the cell nucleus, stained bright magenta and cyan against the purple backdrop of the nucleus, are enlarged in the progeria cell (right) compared to the normal cell (left). Credit: Salk Institute

Hutchinson-Gilford progeria is a very rare genetic disease causing people to age 8 to 10 times faster than the rest of us and leading to an early death. The rare mutation occurs in one of the structural proteins in the cell nucleus, lamin A, but it has been unclear how a single defective protein in the nucleus causes the myriad rapid-aging features seen in the disease.

Initially, Salk Staff Scientist Abigail Buchwalter, first author of the paper, was interested in whether the mutation was making the lamin A protein less stable and shorter lived. After measuring protein turnover in cultured cells from skin biopsies of both progeria sufferers and healthy people, she found that it wasn’t just lamin A that was affected in the disease.

“We analyzed all the proteins of the nucleus and instead of seeing rapid turnover in just mutant lamin A and maybe a few proteins associated with it, we saw a really broad shift in overall protein stability in the progeria cells,” says Buchwalter. “This indicated a change in protein metabolism that we hadn’t expected.”

Along with the rapid turnover of proteins, the team found that the nucleolus, which makes protein-assembling structures called ribosomes, was enlarged in the prematurely aging cells compared to healthy cells.

Even more intriguing, the team found that nucleolus size increased with age in the healthy cells, suggesting that the size of the nucleolus could not only be a useful biomarker of aging, but potentially a target of therapies to counter both premature and normal aging.

The work supports other research that appears in the same issue showing that decreasing protein synthesis extends lifespan in roundworms and mice. The Hetzer lab plans to continue studying how nucleolus size may serve as a reliable biomarker for aging.

“We always assume that aging is a linear process, but we don’t know that for sure,” says Hetzer, who also holds the Jesse and Caryl Philips Foundation Chair. “A biomarker such as this that tracks aging would be very useful, and could open up new ways of studying and understanding aging in humans.”

The work was funded by the National Institutes of Health, the Nomis Foundation, and the Glenn Center for Aging Research.

See the full article here .

Please help promote STEM in your local schools.

Stem Education Coalition

Every cure has a starting point. Like Dr. Jonas Salk when he conquered polio, Salk scientists are dedicated to innovative biological research. Exploring the molecular basis of diseases makes curing them more likely. In an outstanding and unique environment we gather the foremost scientific minds in the world and give them the freedom to work collaboratively and think creatively. For over 50 years this wide-ranging scientific inquiry has yielded life-changing discoveries impacting human health. We are home to Nobel Laureates and members of the National Academy of Sciences who train and mentor the next generation of international scientists. We lead biological research. We prize discovery. Salk is where cures begin.

From Salk: “New method to rapidly map the “social networks” of proteins”

Salk Institute for Biological Studies

June 26, 2017
Office of Communications
Tel: (858) 453-4100
press@salk.edu

Salk scientists improved upon a classic approach to mapping the interactions between proteins.

A new mapping method let researchers discover new links (gray lines) between two groups of plant proteins (yellow and blue) that have a common structure (the BBX domain), suggesting many different combinations of interactions, rather than a few, are involved in coordinating cellular programs like flowering time and circadian rhythm. Credit: Salk Institute.

Salk scientists have developed a new high-throughput technique to determine which proteins in a cell interact with each other. Mapping this network of interactions, or “interactome,” has been slow going in the past because the number of interactions that could be tested at once was limited. The new approach, published June 26 in Nature Methods, lets researchers test millions of relationships between thousands of proteins in a single experiment.

“The power of this new approach is in the ability we now have to scale it up,” says senior author Joseph Ecker, professor and director of Salk’s Genomic Analysis Laboratory and investigator of the Howard Hughes Medical Institute. “This assay has the potential to begin to address questions about fundamental biological interactions that we haven’t been able to address before.”

The interactome of a cell, like a map of social networks, lets scientists see who’s working with who in the world of proteins. This helps them figure out the roles of different proteins and piece together the different players in molecular pathways and processes. If a newly discovered protein interacts with lots of other proteins involved in cellular metabolism, for instance, researchers can deduce that’s a likely role for the new protein and potentially target it for treatments related to metabolic dysfunction.

Until now, researchers have typically relied on standard high-throughput yeast two-hybrid (Y2H) assays to determine the interactions between proteins. The system requires using a single known protein—known as the “bait”—to screen against a pool of “prey” proteins. But finding all the interactions between, for instance, 1,000 proteins, would require 1000 separate experiments to screen once for each bait’s interaction partners.

“Current technologies essentially require that interactions detected in primary screening get retested individually,” says Shelly Trigg, an NSF Graduate Research Fellow at the University of California, San Diego, in the Ecker lab, and first author of the new paper. “That may no longer be necessary with the screening depth this approach achieves.”

In their new method, Ecker, Trigg and their colleagues added a twist to the standard Y2H assay for a much more effective way of measuring the interactome. The genes for two proteins, each on their own circle of DNA, are added to the same cell. If the proteins of interest interact inside the cell, a gene called Cre is activated. When turned on, Cre physically splices the two individual circles of DNA together, thus pairing the genes of interacting proteins together so the team can easily find them through sequencing. The team can generate a massive library of yeast cells—each containing different pairs of proteins by introducing random combinations of genes on circular DNA called plasmids. When cells are positive for a protein interaction, the researchers can use genetic sequencing to figure out what the two proteins interacting are, using new high-throughput DNA sequencing technologies similar to those used for human genome sequencing. This way, they’re no longer limited to testing one “bait” protein at a time, but could test the interactions between all the proteins in a library at once.

Joseph Ecker (courtesy of Salk Institute) and Shelly Trigg (courtesy of Austin Trigg)

Ecker’s group tested the new method, dubbed CrY2H-seq, on all the transcription factors—a large class of proteins—in the plant Arabidopsis.

“When you take 1,800 proteins and test the interactions among them, that’s nearly 4 million combinations,” says Ecker. “We did that ten times in a matter of a month.”

They revealed more than 8,000 interactions among those proteins tested, giving them new insight into which Arabidopsis transcription factors interact with each other. The data, they say, helps answer longstanding questions about whether certain groups of transcription factors have set functions. Some of the poorly understood transcription factors, they found, interact with more well-understood factors that regulate the plant’s response to auxin, a hormone involved in coordinating plant growth.

In the future, the method could be scaled up to test larger sets of proteins—human cells, for instance, contain about 20,000 different proteins. This easier and faster method to determine the entire interactome of a cell also opens up the possibility of studying how the interactome changes under different conditions—an experiment that’s never been possible in the past.

Other researchers on the study were Renee Garza, Andrew MacWilliams, Joseph Nery, Anna Bartlett, Rosa Castanon, Adeline Goubil, Joseph Feeney, Ronan O’Malley, Shao-shan Carol Huang, Zhuzhu Zhang, and Mary Galli of the Salk Institute.

See the full article here .

Please help promote STEM in your local schools.

Stem Education Coalition

Every cure has a starting point. Like Dr. Jonas Salk when he conquered polio, Salk scientists are dedicated to innovative biological research. Exploring the molecular basis of diseases makes curing them more likely. In an outstanding and unique environment we gather the foremost scientific minds in the world and give them the freedom to work collaboratively and think creatively. For over 50 years this wide-ranging scientific inquiry has yielded life-changing discoveries impacting human health. We are home to Nobel Laureates and members of the National Academy of Sciences who train and mentor the next generation of international scientists. We lead biological research. We prize discovery. Salk is where cures begin.

From U Washington: “Feedback from 1000s of designs could transform protein engineering”

University of Washington

07.12.2017
Leila Gray
206.685.0381
leilag@uw.edu

A model of a computationally designed mini-protein from a large-scale study to test structural stability. Institute for Protein Design.

The stage is set for a new era of data-driven protein molecular engineering as advances in DNA synthesis technology merge with improvements in computational design of new proteins.

This week’s Science reports the largest-scale testing of folding stability for computationally designed proteins, made possible by a new high-throughput approach.

The scientists are from the UW Medicine Institute for Protein Design at the University of Washington in Seattle and the University of Toronto in Ontario.

The lead author of the paper is Gabriel Rocklin, a postdoctoral fellow in biochemistry at the University of Washington School of Medicine. The senior authors are Cheryl Arrowsmith, of the Princess Margaret Cancer Center, the Structural Genomics Consortium and the Department of Medical Biophysics at the University of Toronto, and David Baker, UW professor of biochemistry and a Howard Hughes Medical Institute investigator.

Proteins are biological workhorses. Researchers want to build new molecules, not found naturally, that can perform tasks in preventing or treating disease, in industrial applications, in energy production, and in environmental cleanups.

“However, computationally designed proteins often fail to form the folded structures that they were designed to have when they are actually tested in the lab,” Rocklin said.

In the latest study, the researchers tested more than 15,000 newly designed mini-proteins that do not exist in nature to see whether they form folded structures. Even major protein design studies in the past few years have generally examined only 50 to 100 designs.

“We learned a huge amount at this new scale, but the taste has given us an even larger appetite,” said Rocklin. “We’re eager to test hundreds of thousands of designs in the next few years.”

The most recent testing led to the design of 2,788 stable protein structures and could have many bioengineering and synthetic biology applications. Their small size may be advantageous for treating diseases when the drug needs to reach the inside of a cell.

Design model structures from a comprehensive mutational analysis of stability in natural and designed proteins. UW Institute for Protein Design.

Proteins are made of amino acid chains with specific sequences, and natural protein sequences are encoded in cellular DNA. These chains fold into 3-dimensional conformations. The sequence of the amino acids in the chain guide where it will bend and twist, and how parts will interact to hold the structure together.

For decades, researchers have studied these interactions by examining the structures of naturally occurring proteins. However, natural protein structures are typically large and complex, with thousands of interactions that collectively hold the protein in its folded shape. Measuring the contribution of each interaction becomes very difficult.

The scientists addressed this problem by computationally designing their own, much simpler proteins. These simpler proteins made it easier to analyze the different types of interactions that hold all proteins in their folded structures.

“Still, even simple proteins are so complicated that it was important to study thousands of them to learn why they fold,” Rocklin said. “This had been impossible until recently, due to the cost of DNA. Each designed protein requires its own customized piece of DNA so that it can be made inside a cell. This has limited previous studies to testing only tens of designs.”

To encode their designs of short proteins in this project, the researchers used what is called DNA oligo library synthesis technology. It was originally developed for other laboratory protocols, such as large gene assembly. One of the companies that provided their DNA is CustomArray in Bothell, Wash. They also used DNA libraries made by Agilent in Santa Clara, Calif., and Twist Bioscience in San Francisco.

By repeating the cycle of computation and experimental testing over several iterations, the researchers learned from their design failures and progressively improved their modeling. Their design success rate rose from 6 percent to 47 percent. They also produced stable proteins in shapes where all of their first designs failed.

Their large set of stable and unstable mini-proteins enabled them to quantitatively analyze which protein features correlated with folding. They also compared the stability of their designed proteins to similarly sized, naturally occurring proteins.

The most stable natural protein the researchers identified was a much-studied protein from the bacteria Bacillus stearothermophilus.

The researchers compared the stability of some of their designed proteins to a natural protein found in a bacteria that withstands the high temperatures of hot springs like those in Yellowstone. Alice C. Gray.

This organism basks in high temperatures, like those in hot springs and ocean thermal vents. Most proteins lose their folded structures under such high temperature conditions. Organisms that thrive there have evolved highly stable proteins that stay folded even when hot.

“A total of 774 designed proteins had higher stability scores than this most protease-resistant monomeric protein,” the researchers noted. Proteases are enzymes that break down proteins, and were essential tools the researchers used to measure stability for their thousands of proteins.

The researchers predict that, as DNA synthesis technology continues to improve, high-throughput protein design will become possible for larger, more complex protein structures.

“We are moving away from the old style of protein design, which was a mix of computer modeling, human intuition, and small bits of evidence about what worked before.” Rocklin said. “Protein designers were like master craftsmen who used their experience to hand-sculpt each piece in their workshop. Sometimes things worked, but when they failed it was hard to say why. Our new approach lets us collect an enormous amount of data on what makes proteins stable. This data can now drive the design process.”

Their study was supported by the Howard Hughes Medical Institute and the Natural Sciences and Research Council of Canada. Rocklin is a Merck Fellow of the Life Sciences Research Foundation. Arrowsmith holds a Canadian Research Chair in Structural Genomics.

This work was facilitated by the Hyak supercomputer at the University of Washington and by donations of computing time from Rosetta@home participants.

Rosetta@home project, a project running on BOINC software from UC Berkeley

Dr. David Baker, Baker Lab, U Washington

Hyak supercomputer at the University of Washington

See the full article here .

Please help promote STEM in your local schools.

Stem Education Coalition

The University of Washington is one of the world’s preeminent public universities. Our impact on individuals, on our region, and on the world is profound — whether we are launching young people into a boundless future or confronting the grand challenges of our time through undaunted research and scholarship. Ranked number 10 in the world in Shanghai Jiao Tong University rankings and educating more than 54,000 students annually, our students and faculty work together to turn ideas into impact and in the process transform lives and our world. For more about our impact on the world, every day.
So what defines us —the students, faculty and community members at the University of Washington? Above all, it’s our belief in possibility and our unshakable optimism. It’s a connection to others, both near and far. It’s a hunger that pushes us to tackle challenges and pursue progress. It’s the conviction that together we can create a world of good. Join us on the journey.