PRESS-NEWS.org - Press Release Distribution
PRESS RELEASES DISTRIBUTION

Stanford researchers develop tool to drastically speed up the study of enzymes

Stanford researchers develop tool to drastically speed up the study of enzymes
2021-07-22
(Press-News.org) For much of human history, animals and plants were perceived to follow a different set of rules than the rest of the universe. In the 18th and 19th centuries, this culminated in a belief that living organisms were infused by a non-physical energy or "life force" that allowed them to perform remarkable transformations that couldn't be explained by conventional chemistry or physics alone.

Scientists now understand that these transformations are powered by enzymes - protein molecules comprised of chains of amino acids that act to speed up, or catalyze, the conversion of one kind of molecule (substrates) into another (products). In so doing, they enable reactions such as digestion and fermentation - and all of the chemical events that happen in every one of our cells - that, left alone, would happen extraordinarily slowly.

"A chemical reaction that would take longer than the lifetime of the universe to happen on its own can occur in seconds with the aid of enzymes," said Polly Fordyce, an assistant professor of bioengineering and of genetics at Stanford University.

While much is now known about enzymes, including their structures and the chemical groups they use to facilitate reactions, the details surrounding how their forms connect to their functions, and how they pull off their biochemical wizardry with such extraordinary speed and specificity are still not well understood.

A new technique, developed by Fordyce and her colleagues at Stanford and detailed this week in the journal Science, could help change that. Dubbed HT-MEK -- short for High-Throughput Microfluidic Enzyme Kinetics -- the technique can compress years of work into just a few weeks by enabling thousands of enzyme experiments to be performed simultaneously. "Limits in our ability to do enough experiments have prevented us from truly dissecting and understanding enzymes," said study co-leader Dan Herschlag, a professor of biochemistry at Stanford's School of Medicine.

By allowing scientists to deeply probe beyond the small "active site" of an enzyme where substrate binding occurs, HT-MEK could reveal clues about how even the most distant parts of enzymes work together to achieve their remarkable reactivity.

"It's like we're now taking a flashlight and instead of just shining it on the active site we're shining it over the entire enzyme," Fordyce said. "When we did this, we saw a lot of things we didn't expect." Enzymatic tricks

HT-MEK is designed to replace a laborious process for purifying enzymes that has traditionally involved engineering bacteria to produce a particular enzyme, growing them in large beakers, bursting open the microbes and then isolating the enzyme of interest from all the other unwanted cellular components. To piece together how an enzyme works, scientists introduce intentional mistakes into its DNA blueprint and then analyze how these mutations affect catalysis.

This process is expensive and time consuming, however, so like an audience raptly focused on the hands of a magician during a conjuring trick, researchers have mostly limited their scientific investigations to the active sites of enzymes. "We know a lot about the part of the enzyme where the chemistry occurs because people have made mutations there to see what happens. But that's taken decades," Fordyce said.

But as any connoisseur of magic tricks knows, the key to a successful illusion can lie not just in the actions of the magician's fingers, but might also involve the deft positioning of an arm or the torso, a misdirecting patter or discrete actions happening offstage, invisible to the audience. HT-MEK allows scientists to easily shift their gaze to parts of the enzyme beyond the active site and to explore how, for example, changing the shape of an enzyme's surface might affect the workings of its interior.

"We ultimately would like to do enzymatic tricks ourselves," Fordyce said. "But the first step is figuring out how it's done before we can teach ourselves to do it." Enzyme experiments on a chip

HT-MEK combines two existing technologies to rapidly speed up enzyme analysis. The first is microfluidics, which involves molding polymer chips to create microscopic channels for the precise manipulation of fluids. "Microfluidics shrinks the physical space to do these fluidic experiments in the same way that integrated circuits reduced the real estate needed for computing," Fordyce said. "In enzymology, we are still doing things in these giant liter-sized flasks. Everything is a huge volume and we can't do many things at once."

The second is cell-free protein synthesis, a technology that takes only those crucial pieces of biological machinery required for protein production and combines them into a soupy extract that can be used to create enzymes synthetically, without requiring living cells to serve as incubators.

"We've automated it so that we can use printers to deposit microscopic spots of synthetic DNA coding for the enzyme that we want onto a slide and then align nanoliter-sized chambers filled with the protein starter mix over the spots," Fordyce explained.

Because each tiny chamber contains only a thousandth of a millionth of a liter of material, the scientists can engineer thousands of variants of an enzyme in a single device and study them in parallel. By tweaking the DNA instructions in each chamber, they can modify the chains of amino acid molecules that comprise the enzyme. In this way, it's possible to systematically study how different modifications to an enzyme affects its folding, catalytic ability and ability to bind small molecules and other proteins.

When the team applied their technique to a well-studied enzyme called PafA, they found that mutations well beyond the active site affected its ability to catalyze chemical reactions -- indeed, most of the amino acids, or "residues," making up the enzyme had effects.

The scientists also discovered that a surprising number of mutations caused PafA to misfold into an alternate state that was unable to perform catalysis. "Biochemists have known for decades that misfolding can occur but it's been extremely difficult to identify these cases and even more difficult to quantitatively estimate the amount of this misfolded stuff," said study co-first author Craig Markin, a research scientist with joint appointments in the Fordyce and Herschlag labs.

"This is one enzyme out of thousands and thousands," Herschlag emphasized. "We expect there to be more discoveries and more surprises." Accelerating advances

If widely adopted, HT-MEK could not only improve our basic understanding of enzyme function, but also catalyze advances in medicine and industry, the researchers say. "A lot of the industrial chemicals we use now are bad for the environment and are not sustainable. But enzymes work most effectively in the most environmentally benign substance we have -- water," said study co-first author Daniel Mokhtari, a Stanford graduate student in the Herschlag and Fordyce labs.

HT-MEK could also accelerate an approach to drug development called allosteric targeting, which aims to increase drug specificity by targeting beyond an enzyme's active site. Enzymes are popular pharmaceutical targets because of the key role they play in biological processes. But some are considered "undruggable" because they belong to families of related enzymes that share the same or very similar active sites, and targeting them can lead to side effects. The idea behind allosteric targeting is to create drugs that can bind to parts of enzymes that tend to be more differentiated, like their surfaces, but still control particular aspects of catalysis. "With PafA, we saw functional connectivity between the surface and the active site, so that gives us hope that other enzymes will have similar targets," Markin said. "If we can identify where allosteric targets are, then we'll be able to start on the harder job of actually designing drugs for them."

The sheer amount of data that HT-MEK is expected to generate will also be a boon to computational approaches and machine learning algorithms, like the Google-funded AlphaFold project, designed to deduce an enzyme's complicated 3D shape from its amino acid sequence alone. "If machine learning is to have any chance of accurately predicting enzyme function, it will need the kind of data HT-MEK can provide to train on," Mokhtari said.

Much further down the road, HT-MEK may even allow scientists to reverse-engineer enzymes and design bespoke varieties of their own. "Plastics are a great example," Fordyce said. "We would love to create enzymes that can degrade plastics into nontoxic and harmless pieces. If it were really true that the only part of an enzyme that matters is its active site, then we'd be able to do that and more already. Many people have tried and failed, and it's thought that one reason why we can't is because the rest of the enzyme is important for getting the active site in just the right shape and to wiggle in just the right way."

Herschlag hopes that adoption of HT-MEK among scientists will be swift. "If you're an enzymologist trying to learn about a new enzyme and you have the opportunity to look at 5 or 10 mutations over six months or 100 or 1,000 mutants of your enzyme over the same period, which would you choose?" he said. "This is a tool that has the potential to supplant traditional methods for an entire community."

INFORMATION:

Fordyce is a member of Stanford Bio-X and the Wu Tsai Neurosciences Institute, and an executive committee member of Stanford ChEM-H. Herschlag is member of Bio-X and the Stanford Cancer Institute, and a faculty fellow of ChEM-H. Other Stanford co-authors include Fanny Sunden, Mason Appel, Eyal Akiva, Scott Longwell and Chiara Sabatti.

The research was funded by Stanford Bio-X, Stanford ChEM-H, the Stanford Medical Scientist Training Program, the National Institutes of Health, the Joint Initiative for Metrology in Biology, the Gordon and Betty Moore Foundation, the Alfred P. Sloan Foundation, the Chan Zuckerberg Biohub and the Canadian Institutes of Health Research.


[Attachments] See images for this press release:
Stanford researchers develop tool to drastically speed up the study of enzymes

ELSE PRESS RELEASES FROM THIS DATE:

Alpha variant spread via 'super-seeding' event in UK: Oxford research

2021-07-22
The rapid spread of the Alpha variant of COVID-19 resulted from biological changes in the virus and was enhanced by large numbers of infected people 'exporting' the variant to multiple parts of the UK, in what the researchers call a 'super-seeding' event. Results of the largest phylogeographic analysis ever conducted, published today in the journal Science, maps the spread of the variant (also known as lineage B.1.1.7) from its origins in Kent and Greater London in November 2020 to all but five counties in Wales, Scotland, Northern Ireland and England by 19 January. Dr ...

Soft skin patch could provide early warning for strokes, heart attacks

Soft skin patch could provide early warning for strokes, heart attacks
2021-07-22
Engineers at the University of California San Diego developed a soft and stretchy ultrasound patch that can be worn on the skin to monitor blood flow through major arteries and veins deep inside a person's body. Knowing how fast and how much blood flows through a patient's blood vessels is important because it can help clinicians diagnose various cardiovascular conditions, including blood clots; heart valve problems; poor circulation in the limbs; or blockages in the arteries that could lead to strokes or heart attacks. The new ultrasound patch developed at UC San Diego can continuously monitor ...

California's carbon mitigation efforts may be thwarted by climate change itself

Californias carbon mitigation efforts may be thwarted by climate change itself
2021-07-22
Irvine, Calif., July 22, 2021 - To meet an ambitious goal of carbon neutrality by 2045, California's policymakers are relying in part on forests and shrublands to remove CO2 from the atmosphere, but researchers at the University of California, Irvine warn that future climate change may limit the ecosystem's ability to perform this service. In a paper published today in the American Geophysical Union journal AGU Advances, the UCI Earth system scientists stressed that rising temperatures and uncertain precipitation will cause a decrease in California's natural carbon storage capacity of as much as 16 percent under an extreme climate projection and of nearly 9 percent under a more moderate scenario. "This work highlights the conundrum ...

Cell-analysis technique could combat tuberculosis

2021-07-22
ITHACA, N.Y. - A new method that analyzes how individual immune cells react to the bacteria that cause tuberculosis could pave the way for new vaccine strategies against this deadly disease, and provide insights into fighting other infectious diseases around the world. The cutting-edge technologies were developed in the lab of Dr. David Russell, the William Kaplan Professor of Infection Biology in the Department of Microbiology and Immunology in the College of Veterinary Medicine, and detailed in new research published in the Journal of Experimental Medicine on July 22. For years, Russell's lab has sought to unravel how Mycobacterium tuberculosis (Mtb), the bacteria that cause tuberculosis, infect and persist in their host cells, which are typically ...

New study provides clues to decades-old mystery about cell movement

New study provides clues to decades-old mystery about cell movement
2021-07-22
A new study, led by University of Minnesota Twin Cities engineering researchers, shows that the stiffness of protein fibers in tissues, like collagen, are a key component in controlling the movement of cells. The groundbreaking discovery provides the first proof of a theory from the early 1980s and could have a major impact on fields that study cell movement from regenerative medicine to cancer research. The research is published in the Proceedings of the National Academy of Sciences of the United States of America (PNAS), a peer-reviewed, multidisciplinary, high-impact scientific journal. Directed cell movement, or what scientists call "cell contact guidance," refers to a phenomenon when the orientation of cells ...

Study points to remotely supervised exercise classes as best option during lockdown

2021-07-22
Researchers at the University of São Paulo (USP) in Brazil investigated the effects of regular exercise on the physical and mental health of 344 volunteers during the pandemic. The study compared the effectiveness of three techniques: sessions led in person by a fitness instructor, sessions featuring an online instructor but no supervision, and sessions supervised remotely by an instructor via video call. The two kinds of session with professional supervision had the strongest effects on physical and mental health. According to the researchers, this was due to the possibility of increasing ...

Possible link between late-term births and better academic outcomes, study suggests

2021-07-22
New Brunswick, NJ--Even at term, gestational age may have an impact on children's academic performance, findings of a new study suggest. The research showed an association between gestational age at term and above-average rankings in a number of academic subjects. The study, published in Pediatrics, compared teacher-reported outcomes for 1,405 9-year-old children in the United States, analyzing performance in mathematics, science and social studies, and language and literacy, for those born at 37 through 41 weeks gestation. It found that longer gestational age was significantly associated with average or above-average rankings in all areas. It also suggested a general pattern of worse outcomes for children born at early term (37-38 weeks) and better outcomes for those born at late ...

Eco-friendly plastic from cellulose and water

Eco-friendly plastic from cellulose and water
2021-07-22
Plastics offer many benefits to society and are widely used in our daily life: they are lightweight, cheap and adaptable. However, the production, processing and disposal of plastics are simply not sustainable, and pose a major global threat to the environment and human health. Eco-friendly processing of reusable and recyclable plastics derived from plant-based raw materials would be an ideal solution. So far, the technological challenges have proved too great. However, researchers at the University of Göttingen have now found a sustainable method - "hydrosetting", which uses water at normal conditions - to process and reshape a new type of hydroplastic polymer called cellulose cinnamate (CCi). The research was published ...

Pathogens get comfy in designer goo

Pathogens get comfy in designer goo
2021-07-22
HOUSTON -- (July 22, 2021) -- Researchers who want bacteria to feel right at home in the laboratory have put out a new welcome mat. Rice University bioengineers and Baylor College of Medicine scientists looking for a better way to mimic intestinal infections that cause diarrhea and other diseases have built and tested a set of hydrogel-based platforms to see if they could make both transplanted cells and bacteria comfy. As a mechanical model of intestinal environments, the lab's soft, medium and hard polyethylene glycol (PEG) hydrogels were far more welcoming to the cells that normally line the gut than the glass and plastic usually used by laboratories. These cells can then host bacteria like Escherichia coli that are sometimes pathogenic. The ability to study their ...

What makes a market transaction morally repugnant?

2021-07-22
Many people find it morally impermissible to put kidneys, children, or doctorates on the free market. But what makes a market transaction morally repugnant in the eyes of the public? And which transactions trigger the strongest collective disapproval? Researchers from the Max Planck Institute for Human Development and the Robert Koch Institute have addressed these questions. Their findings, published in Cognition, offer new entry points for policy interventions. Would you be willing to sell a kidney or be paid to spend time on a date? If not, then ...

LAST 30 PRESS RELEASES:

Modesty and boastfulness – perception depends on usual performance

Do sweeteners increase your appetite? New evidence from randomised controlled trial says no 

Women with obesity do not need to gain weight during pregnancy, new study suggests

Individuals with multiple sclerosis face substantially greater risk of hospitalisation and death from COVID-19, despite high rates of vaccination

Study shows obesity in childhood associated with a more than doubling of risk of developing multiple sclerosis in early adulthood

Rice Emerging Scholars Program receives $2.5M NSF grant to boost STEM education

Virtual rehabilitation provides benefits for stroke recovery

Generative AI develops potential new drugs for antibiotic-resistant bacteria

Biofuels could help island nations survive a global catastrophe, study suggests

NJIT research team discovering how fluids behave in nanopores with NSF grant

New study shows association of historical housing discrimination and shortfalls in colon cancer treatment

Social media use may help to empower plastic surgery patients

Q&A: How to train AI when you don't have enough data

Wayne State University researchers uncover potential treatment targets for Zika virus-related eye abnormalities

Discovering Van Gogh in the wild: scientists unveil a new gecko species

Small birds spice up the already diverse diet of spotted hyenas in Namibia

Imaging detects transient “hypoxic pockets” in the mouse brain

Dissolved organic matter could be used to track and improve the health of freshwaters

Indoor air quality standards in public buildings would boost health and economy, say international experts

Positive associations between premenstrual disorders and perinatal depression

New imaging method illuminates oxygen's journey in the brain

Researchers discover key gene for toxic alkaloid in barley

New approach to monitoring freshwater quality can identify sources of pollution, and predict their effects

Bidirectional link between premenstrual disorders and perinatal depression

Cell division quality control ‘stopwatch’ uncovered

Vaccine protects cattle from bovine tuberculosis, may eliminate disease

Andrew Siemion to receive the SETI Institute’s 2024 Drake Award

New study shows how the Crimean-Congo hemorrhagic fever virus enters our cells

Neoadjuvant chemotherapy proves effective for locally advanced penile squamous cell carcinoma

Study flips treatment paradigm in bilateral Wilms tumor, shows resistance to chemotherapy may point toward favorable outcomes

[Press-News.org] Stanford researchers develop tool to drastically speed up the study of enzymes