Category: Chemistry

As the executor of life activities, proteins exert their specific biological functions through interactions such as forming protein complexes. The localization effects, crowding effects, and organelle microenvironments within cells are crucial for maintaining the structure and function of protein complexes.

Recently, a research team led by Prof. Zhang Lihua from the Dalian Institute of Chemical Physics (DICP) of the Chinese Academy of Sciences (CAS) has developed a glycosidic-bond-based mass-spectrometry-cleavable cross-linker, which improves the data analysis throughput and identification accuracy of cross-linking information with good amphiphilicity and biocompatibility. It enables in-vivo cross-linking of protein complexes in live cells and achieves large-scale and precise analysis. The study was published in Angewandte Chemie International Edition on March 30.

Chemical cross-linking mass spectrometry (CXMS), especially in-vivo CXMS, is a large-scale analysis of in-situ conformation and interaction interface of protein complexes in living cells. However, in-vivo CXMS in living cells faces challenges such as high cell disturbance and complex spectra retrieval of cross-linked peptides.

In this study, the researchers incorporated glycosidic bonds into the design of functional cross-linkers based on the high biocompatibility of glucose molecules and the mass spectrometry cleavable feature of glycosidic bonds. They screened and obtained trehalose, a highly biocompatible molecule, as the skeleton molecule and developed a mass spectrometry cleavable cross-linker, trehalose disuccinimidyl succinate (TDS).

This cross-linker showed superior cell viability maintenance compared to currently reported membrane-permeable chemical cross-linkers and enabled efficient cross-linking of protein complexes in cells under low disturbance conditions.

The researchers found that low-energy glycosidic bond–high-energy peptide bond mass spectrometry selective fragmentation mode reduced analysis complexity of the cross-linked peptide fragment spectra, significantly improving the efficiency and accuracy of cross-linked peptide identification.

They identified conformation of 1,453 proteins corresponding to more than 3,500 cross-linked peptide pairs, and 843 protein-protein interaction information from Hela cells.

“We have accurately realized in-vivo cross-linking and global analysis of protein complexes in live cells, and provided an important toolkit for exploring the interaction sites of protein function regulation in live cell microenvironment,” said Prof. Zhang.

More information: Jing Chen et al, A Glycosidic‐Bond‐Based Mass‐Spectrometry‐Cleavable Cross‐linker Enables In Vivo Cross‐linking for Protein Complex Analysis, Angewandte Chemie International Edition (2023). DOI: 10.1002/anie.202212860

Journal information: Angewandte Chemie International Edition 

Provided by Chinese Academy of Sciences 

Researchers at SLAC National Accelerator Laboratory captured one of the fastest movements of a molecule called ferricyanide for the first time by combining two ultrafast X-ray spectroscopy techniques. They think their approach could help map more complex chemical reactions like oxygen transportation in blood cells or hydrogen production using artificial photosynthesis.

The research team from SLAC, Stanford and other institutions started with what is now a fairly standard technique: They zapped a mixture of ferricyanide and water with an ultraviolet laser and bright X-rays generated by the Linac Coherent Light Source (LCLS) X-ray free-electron laser. The ultraviolet light kicked the molecule into an excited state while the X-rays probed the sample’s atoms, revealing features of ferricyanide’s atomic and electronic structure and motion.

What was different this time is how the researchers extracted information from the X-ray data. Instead of studying only one spectroscopic region, known as the Kβ main emission line, the team captured and analyzed a second emission region, called valence-to-core, which has been significantly more challenging to measure on ultrafast timescales. Combining information from both regions enabled the team to obtain a detailed picture of the ferricyanide molecule as it evolved into a key transitional state.

The team showed that ferricyanide enters an intermediate, excited state for about 0.3 picoseconds—or less than a trillionth of a second—after being hit with a UV laser. The valence-to-core readings then revealed that following this short-lived, excited period, ferricyanide loses one of its molecular cyanide “arms,” called a ligand. Ferricyanide then either fills this missing joint with the same carbon-based ligand or, less likely, a water molecule.

“This ligand exchange is a basic chemical reaction that was thought to occur in ferricyanide, but there was no direct experimental evidence of the individual steps in this process,” SLAC scientist and first author Marco Reinhard said. “With only a Kβ main emission line analysis approach, we wouldn’t really be able to see what the molecule looks like when it is changing from one state to the next; we’d only obtain a clear picture of the beginning of the process.”

“You want to be able to replicate what nature does to improve technology and increase our foundational scientific knowledge,” SLAC senior scientist Dimosthenis Sokaras said. “And in order to better replicate natural processes, you have to know all of the steps, from the most obvious to those that happen in the dark, so to speak.”

In the future, the research team wants to study more complex molecules, such as hemeproteins, which transport and store oxygen in red blood cells—but which can be tricky to study because scientists do not understand all the intermediate steps of their reactions, Sokaras said.

The research team refined their X-ray spectroscopy technique at SLAC’s Stanford Synchrotron Radiation Lightsource (SSRL) and LCLS over many years, and then combined all this expertise at LCLS’s X-ray Correlation Spectroscopy (XCS) instrument to capture the molecular structural changes of ferricyanide. The team published their results today in Nature Communications.

“We leveraged both SSRL and LCLS to complete the experiment. We couldn’t have finished developing our method without access to both facilities and our longstanding collaboration together,” said Roberto Alonso-Mori, SLAC lead scientist. “For years, we have been developing these methods at these two X-ray sources, and now we plan to use them to uncover previously inaccessible secrets of chemical reactions.”

More information: Marco Reinhard et al, Ferricyanide photo-aquation pathway revealed by combined femtosecond Kβ main line and valence-to-core x-ray emission spectroscopy, Nature Communications (2023). DOI: 10.1038/s41467-023-37922-x

Journal information: Nature Communications 

Provided by SLAC National Accelerator Laboratory 

Water oxidation reaction involves a four-electron and four-proton transfer process, which requires an uphill energy transformation and limits the efficiency of the overall photocatalytic water splitting reaction.

Although loading appropriate water oxidation cocatalysts can enhance the performance of water oxidation reactions, the interfacial barrier between the semiconductor and the water oxidation cocatalyst can impede the transfer and utilization of photogenerated charges.

Recently, a research team led by Profs. Li Can and Li Rengui from the Dalian Institute of Chemical Physics (DICP) of the Chinese Academy of Sciences (CAS) has developed a strategy to controllably assemble a charge-transfer mediator in photocatalysis, which could increase surface charge-transfer efficiency and photocatalytic water oxidation activity. The study was published in Angewandte Chemie International Edition on March 23.

Inspired by natural photosynthesis, the researchers employed partially oxidized graphene (pGO) as a charge-transferring mediator on the hole-accumulating facets of lead chromate (PbCrO₄) photocatalyst. The pGO could be selectively assembled on the hole-accumulating facets of PbCrO₄ by an ultrasonic deposition process, and cobalt-complex Co₄O₄ molecules could be anchored on the pGO as water oxidation cocatalyst.

Based on techniques such as surface photovoltage spectroscopy, they confirmed that introducing the pGO charge transfer mediator between the hole-accumulation facets of PbCrO₄ and Co₄O₄ molecules could effectively suppress charge recombination at the interface, thus prolonging the lifetime of photogenerated charges and enhancing photocatalytic water oxidation performance.

“The strategy of rationally assembling charge transfer mediator provides a feasible way for accelerating charge transfer and charge utilization in semiconductor photocatalysis,” said Prof. Li Rengui.

More information: Wenchao Jiang et al, Graphene Mediates Charge Transfer between Lead Chromate and a Cobalt Cubane Cocatalyst for Photocatalytic Water Oxidation, Angewandte Chemie International Edition (2023). DOI: 10.1002/anie.202302575

Journal information: Angewandte Chemie International Edition 

Provided by Chinese Academy of Sciences 

A small team of chemists at the Russian Academy of Sciences, has found that metal atoms, not nanoparticles, play the key role in catalysts used in fine organic synthesis. In the study, reported in the Journal of the American Chemical Society, the group used multiple types of electron microscopy to track a region of a catalyst during a reaction to learn more about how it was proceeding.

Prior research has shown that there are two main methods for studying a reaction. The first is the most basic: As ingredients are added, the reaction is simply observed and/or measured. This can be facilitated through use of high-speed cameras. This approach will not work with nanoscale reactions, of course. In such cases, chemists use a second method: They attempt to capture the state of all the components before and after the reaction and then compare them to learn more about what happened.

This second approach leaves much to be desired, however, as there is no way to prove that the objects under study correspond with one another. In recent years, chemists have been working on a new approach: Following the action of a single particle during the reaction. This new method has proven to have merit but it has limitations as well—it also cannot be used for reactions that occur in the nanoworld. In this new effort, the researchers used multiple types of electron microscopy coupled with machine-learning algorithms.

To test their ideas, the researchers used a carbon substrate with embedded palladium nanoparticles as a catalyst. By studying reactions using such a catalyst with several types of electron microscopes and then training machine learning algorithm with the results, they were able to track a region of the catalyst as it moved through a reaction. They were able to see that there were individual metal atoms as well as clusters in addition to the nanoparticles playing a role in the reaction. Further study showed that approximately 99% of catalytic activity was due to the palladium atoms, rather than the nanoparticles despite them making up just 1% of the palladium mass.

More information: Alexey S. Galushko et al, Time-Resolved Formation and Operation Maps of Pd Catalysts Suggest a Key Role of Single Atom Centers in Cross-Coupling, Journal of the American Chemical Society (2023). DOI: 10.1021/jacs.3c00645

Journal information: Journal of the American Chemical Society 

© 2023 Science X Network

University of Toronto researchers are investigating exposure to second-hand—and even third-hand—marijuana smoke in homes, including the THC that can collect on floors and surfaces.

The researchers, in Faculty of Applied Science & Engineering, have published a new study that models how THC—the main psychoactive ingredient in cannabis—behaves and transforms once it is released in an indoor environment. The study is published in the journal Environmental Science: Atmospheres.

The model enables researchers to explore mitigation strategies that could reduce involuntary exposure levels.

“We began our research on tetrahydrocannabinol (THC), which is the psychoactive part of cannabis that causes intoxication, because when we looked at second- and third-hand smoke, we started to see how much involuntary exposure happens,” says Amirashkan Askari, a Ph.D. candidate in department of chemical engineering and applied chemistry.

Askari co-authored the study with U of T Engineering Associate Professor Arthur Chan and Frank Wania, a professor in the department of physical and environmental sciences at U of T Scarborough.

Between April 2021 and March 2022, Canadians spent $4 billion on regulated, adult-use cannabis, according to Statistics Canada. Dried cannabis accounted for 71.1 percent of sales, indicating that smoking is the most popular method of consumption.

“Any type of smoking, whether it is tobacco or cannabis, leaves behind a suite of pollutants that can remain in homes,” says Chan. “We now have sufficient chemical knowledge about THC to model its behavior in a typical indoor environment.”

Moreover, involuntary THC exposure can continue long after smoking has ceased. This is due to THC’s large and complex chemical structure, which has a strong tendency to stick to surfaces and create third-hand exposure,” says Askari.

“There are a lot of surfaces indoors—tables, chairs and floors. When you calculate the ratio of surfaces to volume, it is quite elevated compared to the outdoors,” he says. “So, when a pollutant is emitted, it always has the chance to migrate from air to surfaces.

“Involuntary exposure to pollutants starts to become more important when we consider infants and children who reside in homes where this smoking takes place. Children tend to touch surfaces more than adults as they crawl or play; they are also known to frequently put their hands or objects in their mouth.”

Askari used a time-dependent indoor mass-balance model to forecast the level of human exposure to THC. The study also examined the effectiveness of mitigating strategies—from air purifiers to surface cleaners—in reducing second- and third-hand exposure from marijuana smoke.

The model was run for one simulated year under the assumption that THC from single-stream smoke (the lighted end) of a burning cannabis cigarette was emitted into the indoor air for one hour daily.

By modeling the exposure level of an adult and a toddler (who were distinguished by body weight) Askari predicted that residents of all ages who are present during smoking sessions are vulnerable to high levels of involuntary second-hand THC.

The exposure analysis also found that carpet and flooring materials were significant reservoirs of THC that migrated from air to surface. Since younger children are prone to object mouthing—a common part of infant and toddler development—this makes them especially sensitive to THC from third-hand exposure. These results, the study concludes, highlight the importance of preventing children from accessing spaces where cannabis smoking takes place, both during and after smoking.

“When it comes to improving indoor air quality, the best way to degrade air pollutants is to shut down the source,” says Askari. “But if our aim is to suppress it, we found the most effective measures were strategies that target the air particles directly. So, if you have an air purifier unit that filters particulate matter from the air, that will reduce that exposure significantly.”

While the researchers’ initial study used computer simulation, the second phase of this cannabis and indoor air pollution research involves experiments in collaboration with the Centre for Addiction and Mental Health (CAMH).

“We had volunteers come in and either smoke or vape cannabis,” says Askari. “We measured the composition of air in real time—while they were consuming the cannabis—so we could see what happens to the air quality. We also did comparisons between smoking and vaping.”

The results from this second study have not been published, but the team hopes this research will help individuals and policymakers better understand how this source of indoor air pollution impacts the health of communities.

“We hope that people will start paying more attention to indoor air quality, not just during these high-emitting activities, but also long after they are over,” Chan says. “Keeping our homes well-ventilated is very effective at lowering our exposures, even if it is just for a brief period of time during and after smoking.”

More information: Amirashkan Askari et al, Modeling the fate and involuntary exposure to tetrahydrocannabinol emitted from indoor cannabis smoking, Environmental Science: Atmospheres (2023). DOI: 10.1039/D2EA00155A

Provided by University of Toronto 

People’s ability to regenerate bones declines with age and is further decreased by diseases such as osteoporosis. To help the aging population, researchers are looking for new therapies that improve bone regeneration.

Now, an interdisciplinary team of researchers from the Biotechnology Center (BIOTEC) and the Medical Faculty of TU Dresden along with a group from Max Bergmann Center of Biomaterials (MBC) developed novel bio-inspired molecules that enhance bone regeneration in mice. The results were published in the journal Biomaterials.

As people age, their ability to regenerate bones decreases. Fractures take longer to heal and diseases like osteoporosis only add to it. This represents a serious health challenge to the aging population and an increasing socioeconomic burden for the society. To help combat this issue, researchers are looking for new therapeutic approaches that can improve bone regeneration.

A team of scientists from Dresden used computer modeling and simulations to design novel bio-inspired molecules to enhance bone regeneration in mice. The new molecules can be incorporated into biomaterials and applied locally to bone defects. These new molecules are based on glycosaminoglycans, which are long-chained sugars such as hyaluronic acid or heparin.

A sweet solution for an old bone

“Thanks to our group’s work and the work of other researchers, we know a distinct molecular pathway that regulates bone formation and repair. In fact, we can narrow it down to two proteins that work together to block bone regeneration, sclerostin and dickkopf-1” explains Prof. Lorenz Hofbauer, “The big challenge for developing drugs that improve bone healing is to efficiently turn off both of these proteins, which act as brake signals, at the same time.”

An interdisciplinary approach was a key to this challenge. The Structural Bioinformatics group led by Prof. Maria Teresa Pisabarro at the Biotechnology Center (BIOTEC) of TU Dresden and the Functional Biomaterials group led by PD Dr. Vera Hintze at the Max Bergmann Center of Biomaterials (MBC), Institute of Materials Science of TU Dresden combined their know-how with bone expert Prof. Lorenz Hofbauer at the Medical Faculty of TU Dresden.

“For several years, we have harnessed the power of computer simulations to investigate how proteins regulating bone formation interact with their receptors. All this to design new molecules that can efficiently interfere with these interactions. We worked in tandem between the computer and the bench, designing new molecules and testing them, feeding the results back to our molecular models and learning more about the molecular properties required for our goal,” explains Prof. Pisabarro.

Finally, the team of Lorenz Hofbauer’s Bone Lab used a biomaterial loaded with the new molecules on bone defects in mice to test their effectiveness. The group found that materials containing the novel molecules outperformed the standard biomaterial and enhanced bone healing by up to 50%, which indicates their potential for improving bone regeneration.

Value-added chain: From computer to the lab bench and back

The multidisciplinary team used rational drug design to create novel molecules with tailored properties and minimal side effects. By using computational methods to predict and refine the properties of the designed molecules, the team was able to develop a series of candidates with the greatest potential for turning off the proteins that block bone regeneration.

Pisabarro group’s expertise allowed the thorough analysis of the three-dimensional (3D) structures of the two proteins that block bone regeneration. With that, they were able to model their interaction with their receptors in 3D and identify so-called hot spots, i.e., specific physicochemical and dynamic properties that are essential for the biological interaction to occur.

“We used molecular modeling to design new structures that mimic relevant receptor interactions with both proteins. We wanted this binding to be stronger than their natural interactions. In this way, our novel molecules would simultaneously hijack the proteins and effectively turn them off to turn the bone regeneration on,” explains Prof. Pisabarro.

“The molecules designed by Pisabarro’s group were synthesized by our colleagues at the Free University of Berlin and then analyzed regarding their protein binding properties via biophysical interaction analysis,” says PD Dr. Hintze. “For each of the molecules we were able to measure the binding strength with the proteins and their interference with natural receptor binding of the proteins. Thus, we could reveal empirically how effective each of the small molecules could be at turning off the inhibitory proteins.” Hofbauer group then tested the biological relevance of these interaction studies in a cell culture model and later in mice.

The results of such iterative testing are a valuable asset that enhances the current molecular models of the Pisabarro group and can be used to guide the development of novel and better molecules in the future. Such an approach also ensures that animal research is minimized and enters the project only in its final phase.

The team’s findings represent an exciting step forward in preclinical development. The newly designed molecules could potentially be used to turn off the proteins that block bone regeneration and lead to the development of novel, more effective treatments for bone fractures and other bone-related conditions.

More information: Gloria Ruiz-Gómez et al, Rational engineering of glycosaminoglycan-based Dickkopf-1 scavengers to improve bone regeneration, Biomaterials (2023). DOI: 10.1016/j.biomaterials.2023.122105

Journal information: Biomaterials 

Ion-transport membranes are vital components of clean-energy technologies, such as CO₂ electrolyzers, water electrolyzers, fuel cells, redox flow batteries and ion-capture electrodialysis. These membranes must screen out specific substances to prevent crossover while efficiently conducting specific ions.

Polymer materials have the advantages of low cost, manufacturing scalability and small footprint, and thus dominate the use of ion-transport membranes in practical modules. However, the existing polymer membranes suffer from a ubiquitous “conductivity-selectivity” trade-off: highly conductive membranes tend to exhibit low selectivity and vice versa. This trade-off presents a challenge in developing membrane materials that meet the required performance criteria.

In a study published in Nature on April 26, the research team led by Professor Xu Tongwen and Professor Yang Zhengjin from the University of Science and Technology of China (USTC) of the Chinese Academy of Sciences (CAS), and their collaborators, proposed a new type of ion exchange membrane—triazine framework polymer membranes—which can break the conductivity-selectivity trade-off.

Compared to traditional materials, the triazine framework polymer membranes exhibited much enhanced capacity in both anti-swelling and anti-aging, showing an extremely low swelling ratio on water absorption. Their rigid channels ensured high selectivity from size-sieving, thus enabling extremely low permeability of active materials.

With proper control over the chemistry of rigid pore channels, the researchers observed near-frictionless ion flow within the all-rigid triazine framework polymer membrane (SCTF-BP), with the ion diffusion coefficient close to value in water. This is achieved by the robust micropore confinement within the rigid pore channels and multi-interaction between ion and membrane.

These framework membranes exhibited both extremely low permeability of active materials and ultrahigh ion diffusivity, and their advantages were exemplified as ion-conducting membranes in 2,6-dihydroxy anthraquinone / K₄[Fe(CN)₆] aqueous organic redox flow batteries. The membrane delivered a neat area-specific resistance as low as 0.17 Ω cm², and thus enabled stable cell operation at extreme current densities, from 200 to 500 mA cm^-2, with both high energy efficiency and high-capacity utilization.

These data related to energy efficiency and capacity utilization far surpass those for otherwise identical cells assembled with commercial membranes and state-of-the-art ion-sieving membranes.

This work highlights the importance of secondary interactions to develop high-performing ion-transport membranes. The design strategy proposed is believed to be broadly applicable, considering numerous options of organic reactions and functional monomers that can be utilized to construct polymer frameworks, and directs the fit-for-purpose design of membranes according to practical application demand.

More information: Peipei Zuo et al, Near-frictionless ion transport within triazine framework membranes, Nature (2023). DOI: 10.1038/s41586-023-05888-x

Journal information: Nature 

Provided by Chinese Academy of Sciences 

The human body relies heavily on electrical charges. Lightning-like pulses of energy fly through the brain and nerves and most biological processes depend on electrical ions traveling across the membranes of each cell in our body.

These electrical signals are possible, in part, because of an imbalance in electrical charges that exists on either side of a cellular membrane. Until recently, researchers believed the membrane was an essential component to creating this imbalance. But that thought was turned on its head when researchers at Stanford University discovered that similar imbalanced electrical charges can exist between microdroplets of water and air.

Now, researchers at Duke University have discovered that these types of electric fields also exist within and around another type of cellular structure called biological condensates. Like oil droplets floating in water, these structures exist because of differences in density. They form compartments inside the cell without needing the physical boundary of a membrane.

Inspired by previous research demonstrating that microdroplets of water interacting with air or solid surfaces create tiny electrical imbalances, the researchers decided to see if the same was true for small biological condensates. They also wanted to see if these imbalances sparked reactive oxygen, “redox,” reactions like these other systems.

Appearing on April 28 in the journal Chem, their foundational discovery could change the way researchers think about biological chemistry. It could also provide a clue as to how the first life on Earth harnessed the energy needed to arise.

“In a prebiotic environment without enzymes to catalyze reactions, where would the energy come from?” asked Yifan Dai, a Duke postdoctoral researcher working in the laboratory of Ashutosh Chilkoti, the Alan L. Kaganov Distinguished Professor of Biomedical Engineering and Lingchong You, the James L. Meriam Distinguished Professor of Biomedical Engineering.

“This discovery provides a plausible explanation of where the reaction energy could have come from, just as the potential energy that is imparted on a point charge placed in an electric field,” Dai said.

When electric charges jump between one material and another, they can produce molecular fragments that can pair up and form hydroxyl radicals, which have the chemical formula OH. These can then pair again to form hydrogen peroxide (H2O2) in tiny but detectable amounts.

“But interfaces have seldom been studied in biological regimes other than the cellular membrane, which is one of the most essential part of biology,” said Dai. “So we were wondering what might be happening at the interface of biological condensates, that is, if it is an asymmetric system too.”

Cells can build biological condensates to either separate or trap together certain proteins and molecules, either hindering or promoting their activity. Researchers are just beginning to understand how condensates work and what they could be used for.

Because the Chilkoti laboratory specializes in creating synthetic versions of naturally occurring biological condensates, the researchers were easily able to create a test bed for their theory. After combining the right formula of building blocks to create minuscule condensates, with help from postdoctoral scholar Marco Messina in? Christopher J. Chang’s group at the University of California—Berkeley, they added a dye to the system that glows in the presence of reactive oxygen species.

Their hunch was right. When the environmental conditions were right, a solid glow started from the edges of the condensates, confirming that a previously unknown phenomenon was at work. Dai next talked with Richard Zare, the Marguerite Blake Wilbur Professor of Chemistry at Stanford, whose group established the electric behavior of water droplets. Zare was excited to hear about the new behavior in biological systems, and started to work with the group on the underlying mechanism.

“Inspired by previous work on water droplets, my graduate student, Christian Chamberlayne, and I thought that the same physical principles might apply and promote redox chemistry, such as the formation of hydrogen peroxide molecules,” Zare said. “These findings suggest why condensates are so important in the functioning of cells.”

“Most previous work on biomolecular condensates has focused on their innards,” Chilkoti said. “Yifan’s discovery that biomolecular condensates appear to be universally redox-active suggests that condensates did not simply evolve to carry out specific biological functions as is commonly understood, but that they are also endowed with a critical chemical function that is essential to cells.”

While the biological implications of this ongoing reaction within our cells is not known, Dai points to a prebiotic example of how powerful its effects might be. The powerhouses of our cells, called mitochondria, create energy for all of our life’s functions through the same basic chemical process. But before mitochondria or even the simplest of cells existed, something had to provide energy for the very first of life’s functions to begin working.

Researchers have proposed that the energy was provided by thermal vents in the oceans or hot springs. Others have suggested this same redox reaction that occurs in water microdroplets was created by the spray of ocean waves.

But why not condensates instead?

“Magic can happen when substances get tiny and the interfacial volume becomes enormous compared to its volume,” Dai said. “I think the implications are important to many different fields.”

More information: Yifan Dai et al, Interface of biomolecular condensates modulates redox reactions, Chem (2023). DOI: 10.1016/j.chempr.2023.04.001

Journal information: Chem 

Provided by Duke University 

Scripps Research scientists have developed a new strategy for identifying small molecules that can change the function of proteins, offering a promising path for discovering targeted drugs. In collaboration with scientists at other institutions, the group used their new approach to find small molecules that can alter the activity of proteins involved in cancer.

The research, published in Molecular Cell on April 20, improves on previous methods that could screen for whether small molecules selectively attached to proteins, but not whether they affected the proteins’ biological activities. The new method revolves around using two mirror-image versions of a small molecule and comparing how they change the size of protein complexes in cells.

“The ability of small molecules to specifically bind to a protein and cause a biological consequence is the fundamental basis for most drugs today,” says senior author Benjamin Cravatt, Ph.D., Gilula Chair of Chemical Biology at Scripps Research. “With this assay, we’re expanding our ability to discover these small molecules that not only bind proteins, but have functional impacts.”

In recent years, Cravatt’s lab has designed sets of small chemicals that can irreversibly bind to certain parts of proteins. However, screening these chemical libraries to discover their possible impact on protein function was generally a slow and tedious process. Since individual proteins have different roles in cell biology, researchers often have to develop specialized functional screens for each protein of interest. One screen, for instance, might determine whether the chemicals affected cell growth, while another might determine whether the chemicals changed levels of a different molecule.

“Just because a small molecule engages a protein physically doesn’t mean that it changes the protein’s function in the cell,” says co-first author Jarrett Remsberg, Ph.D., who carried out the work as an American Cancer Society postdoctoral research fellow in the Cravatt lab at Scripps Research. Former graduate student Michael Lazear, Ph.D. and postdoctoral fellow Martin Jaeger, Ph.D. were also first authors of the paper.

In the new work, Cravatt’s group used the conglomeration of proteins into complexes as a proxy for their function. Proteins often work by binding to other proteins—if this binding doesn’t happen or if it is induced to happen, it indicates a protein’s function may have changed.

The research team designed pairs of “mirror image” molecules, called stereoisomers, that could each bind irreversibly to proteins in the same way that their previous chemical libraries had worked. The pairs of stereoisomers let them be sure that the impact of each small molecule was due to its unique structure (if only one version of a molecule changes the proteins’ function, it is likely a specific and direct interaction).

Once they exposed cells to the pairs of stereoisomers, they tested whether a protein-of-interest was in a different size complex, using a technique called size exclusion chromatography in which proteins are sifted through beads with different sized pores.

To show the utility of this approach, the researchers screened the set of small molecules for their ability to change the sizes of protein complexes in prostate cancer cells. They pinpointed a molecule, MY-1B, which selectively disrupted a complex of proteins known as PA28, previously found to play a role in degrading proteins in cancer. Further work in leukemia cells confirmed that, by specifically binding to the protein PMSE1, MY-1B or a related compound (but not their mirror images) could effectively inactivate the PA28 complex.

Cravatt and colleagues also followed up on an observation that a different chemical, EV-96, changed the size of a protein complex involved in splicing strands of RNA inside cells. The team discovered that EV-96 slowed the growth of cancer cells and pinpointed SF3B1 as the protein the chemical was binding to.

In both cases, the new chemicals represent the first time scientists have been able to target the protein complexes—PA28 and the so-called spliceosome— with small, simple synthetic chemicals.

“This means that researchers have new chemical tools in their arsenal that they didn’t have before,” says Remsberg. “It’s an opportunity for better understanding these proteins as well as investigating potential therapeutic opportunities.”

The team hopes their approach can be expanded to use other functional readouts than complex size, and they intend to use it to study different cell types in the future.

“The long-term idea is that we can use this approach to discover chemical compounds that impinge upon any readout,” says Cravatt. “There are certainly other readouts that we hope to be able to look at in the future.”

More information: Michael R. Lazear et al, Proteomic discovery of chemical probes that perturb protein complexes in human cells, Molecular Cell (2023). DOI: 10.1016/j.molcel.2023.03.026. www.cell.com/molecular-cell/pd … -2765(23)00239-3.pdf

Journal information: Molecular Cell 

Provided by The Scripps Research Institute 

Hair styling can be a potent form of self-expression, whether it features dramatic updos, intricate braids or crazy colors. Beyond being a reflection of our personality, these strands contain compounds that could one day appear in bandages, sunscreens or other products. Researchers reporting in ACS Omega have now designed a simple, green process to extract both keratin and melanin from human hair for these possible applications without harsh chemicals or excessive waste.

Hair is made up of protein filaments consisting of many different layers and components. Its structure comes from the protein keratin, which can also be found in fingernails, horns and feathers. Its color is provided by melanin, a group of pigment molecules that are also found in the skin and eyes. In addition, melanin has antioxidative properties and can help shield against ultraviolet light.

These qualities make the compounds suitable for biomedical applications; however, since most discarded hair is incinerated or dumped in landfills, its keratin and melanin are largely unused as well. Chemically extracting them from hair is possible, but current protocols either can only extract one compound at a time, or rely on harsh chemicals and complicated steps. So, Paulomi Ghosh and colleagues wanted to develop a straightforward method to extract both keratin and melanin from human hair with a single procedure, using a recyclable, green solvent.

The researchers collected samples of hair from local salons, then washed and cut them into small slices. Then, they mixed the hair with an ionic liquid, which dissolved the mixture by interrupting the hydrogen bonds that held the keratin proteins together. When heated and poured into a hydrochloric acid solution, the melanin pigments precipitated out and were collected. Next, the researchers performed dialysis to collect the keratin proteins. The ionic liquid was recycled and reused in subsequent reactions, without a significant impact on the reaction’s yield.

Recovered keratin was compatible with blood, suggesting that it could be used in heavy-duty hemostatic bandages. This extraction procedure also maintained the natural structure of the melanin, which was lost in other, harsher methods. Because the melanin had good antioxidative and UV shielding properties, the team says it could be used in sun-protective products or films. The researchers say that this technique could serve as a green way to sustainably extract useful biopolymers from otherwise discarded materials.

More information: Ashmita Mukherjee et al, One-Pot Extraction of Bioresources from Human Hair via a Zero-Waste Green Route, ACS Omega (2023). DOI: 10.1021/acsomega.3c01428

Journal information: ACS Omega 

Provided by American Chemical Society