(This article was originally published on EurekAlert! on September 7, 2021)
Scientists from The Hong Kong University of Science and Technology (HKUST) and The Hong Kong Polytechnic University (PolyU) have developed an in-vitro vesicle formation assay, shedding light on cargo clients and factors that mediate vesicular trafficking and providing a robust tool to offer novel insights into the secretory pathway.
The secretory pathway is a very important process that takes place in human cells. Many growth factors, hormones and other important factors in the human body are secreted from cells through the secretory pathway to perform their physiological functions. In addition, many newly synthesized proteins must be transported to specific subcellular localization through the secretory pathways to perform their functions. In the secretory transport pathway, transport vesicles function as vehicles to carry cargo molecules. Similar to logistics and delivery services in our daily lives, the transportation of cargo molecules to the correct target sites depends on whether the cargo molecules are accurately sorted into specific transport vesicles. Defects in cargo sorting would induce defects in establishing cell polarity, immunity, as well as other physiological processes.
The key players that mediate protein sorting in the secretory pathway include small GTPases of the Arf family and cargo adaptors. The Arf family GTPases cycle between a GDP-bound inactive state and a GTP-bound active state. Upon GTP binding, Arf proteins mediate membrane recruitment of various cytosolic cargo adaptors. Once recruited onto the membranes, these cargo adaptors recognize sorting motifs on the cargo proteins to package cargo proteins into vesicles.
Although significant progress has been achieved in understanding the general steps of cargo sorting, the spectrum of cargo clients of a specific Arf family member or cargo adaptors remains largely unexplored. Led by Prof. GUO Yusong, Associate Professor of Division of Life Science at HKUST and Prof. YAO Zhongping from PolyU, the research team used an in-vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry analyses of the isolated vesicles revealed cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner. One of them, FAM84B interacts with cargo adaptors and regulates transport of transmembrane cargo proteins. In addition, they uncovered novel cargo proteins that depend on GTP hydrolysis to be captured into vesicles.
Then they utilized this assay and identified cytosolic proteins that depend on a specific Arf family protein, SAR1A, to be recruited to vesicle membranes. One of the cytosolic protein, PRRC1, is recruited to endoplasmic reticulum (ER) exit sites, and interacts with the inner COPII coat. Its absence increases membrane association of COPII and affects ER-to-Golgi trafficking.
Utilizing this assay, they also identified the clients of COPII vesicles. These clients include two cargo receptors, SURF4 and ERGIC53. Through analyzing the protein composition of vesicles isolated from control cells or cells depleted of SURF4 or ERGIC53, they revealed specific clients of each of these two cargo receptors.
These results indicate that the vesicle formation assay in combination with quantitative mass spectrometry analysis is a robust and powerful tool to systematically reveal cargo proteins that depend on a specific factor to be packaged into vesicles, to analyze protein profiling of transport vesicles under different physiological conditions, and to uncover cytosolic proteins that interact with a specific factor on vesicle membranes.
This study was recently published in Proceedings of the National Academy of Sciences.
Prof. Guo’s research team focuses on investigating molecular mechanisms regulating protein sorting in the secretory pathway. Dr. HUANG Yan (a postdoctoral research associate from Prof. Guo’s lab at HKUST), Dr. YIN Haidi from PolyU and Dr. LI Baiying from CUHK are co-first authors of this study. Mr. LIU Yang, Dr. TANG Xiao, Miss WANG Wo and Miss WU Zhixiao from the Guo lab also participated in this study.
A team of researchers from the Hong Kong University of Science and Technology (HKUST) has developed an inexpensive, lightweight, and non-toxic (lead-free) photo-battery that has dual functions in harvesting solar energy and storing energy on a single device, making it possible to charge a battery under the sun, without having to plug the device into the wall.
The increasing demand for sustainable energy sources has driven a surge of interest in solar energy and developing storage devices for it. One such device, the photo-battery, is capable of both generating and storing energy in a single device architecture. In theory, this design should permit increased energy storage efficiency and energy density, while decreasing ohmic losses, relaxing packaging requirements and thus reducing the weight, the bulk, and the cost of the system. In reality, however, the poor interface between materials tends to create problems with charge transport, greatly reducing the efficiency in comparison to the simple system of a solar cell wired to an external battery.
A team led by Prof. Jonathan Eugene HALPERT, Assistant Professor from the Department of Chemistry at HKUST, has made advancements towards developing more efficient photobatteries by expanding the utility of a class of materials known as perovskite, which has had applications in solar cells and most recently in batteries. The perovskite halide the team developed acts as a photoelectrode that can harvest energy under illumination without the assistance of an external load in a lithium-ion battery, and is in stark contrast with its existing counterpart for it does not contain lead, hence it has higher stability in air and is free from the concerns of lead poisoning. For their research, the team has replaced lead with bismuth (Bi), a non-toxic element, and forming a strongly light-absorbing crystalline material.
The lithium-ion battery works by allowing electrons to move from a high energy state to a lower one, while doing work in an external circuit. The photobattery has a mechanism similar to an ordinary battery except that it need not be supplied current or plugged into the wall to be charged electrically, but can be charged photoelectrically under the sun. The active material in this new battery is the lead-free perovskite which, when put under light, absorbs a photon and generates a pair of charges, known as an electron and a hole. The team conducted chrono-amperometry experiments under light and in dark to analyze the increase in charging current caused by the light, and recorded a photo-conversion efficiency rate of 0.428% on photocharging the battery after the first discharge. The next step of the team is to experiment with different materials for better performance and efficiency, so that the photobattery can be commercialized in the market.
“At present, we plug all our appliances into the wall to charge them. With further development in this field of photobatteries, we might not have to plug them in at all in the future,” said Prof. Halpert. “We might be able to harvest solar energy and use it to fulfil the power requirements of any devices with modest power needs. Our work is one of the initial steps taken in this field, and, of course, a lot of improvements will be needed to achieve better performance, but we are confident that we can improve its stability and average efficiency with further refinement.”
This photobattery can serve as the built-in battery for devices such as smartphones or tablets, and even remote energy storage applications, which can be made easy with these photobatteries for they are lightweight and portable. It should also help lower production cost when compared to a system consisting of a solar cell plus an external battery since only the battery part is required.
This study was recently published in the scientific journal Nano Letters on June 16.
(This article was published on EurekAlert! on August 17, 2021)
Scientists from the Hong Kong University of Science and Technology (HKUST) have recently discovered a non-classical nucleation process that can greatly facilitate ice formation on foreign surfaces. This finding lays the foundation to predict and control crystallization processes.
Ice is omnipresent and profoundly impacts our daily life, influencing areas such as climate change, transportation, and energy consumption. Understanding the process of ice formation can decelerate the rate at which glaciers melt and sea levels rise and alleviate other major environmental concerns. Since ice formation is mainly governed by ice nucleation followed by the growth of the nuclei, scientists have put in a great effort to understand the thermodynamics and kinetics behind the nucleation processes. Ice nucleation can occur in two distinctive ways: homogeneously in bulk water or heterogeneously on the surface of a solid material, where heterogeneous ice nucleation (HIN) is the predominant mode of ice formation on earth. However, unlike homogeneous ice nucleation, the water-surface interactions present in HIN make the nucleation process sensitive to surface properties. Understanding how surfaces impact the nucleation process is a promising approach to better predict and control crystallization processes.
A common model used to quantify nucleation kinetics based on a thermodynamic framework, classical nucleation theory (CNT), suggests that water molecules must form an ice nucleus of critical size before a crystallization process occurs. The formation of the critical ice nucleus is associated with a single free energy barrier, which needs to be overcome to trigger further ice growth. However, over the years, both experiments and simulations have revealed that CNT is often insufficient to describe some complex nucleation processes. Consequently, CNT has been a subject of immense debate, and non-classical nucleation theories have been alternatively proposed.
Different from CNT, which is based on overcoming a single free energy barrier, non-classical nucleation theories suggest that nucleation processes consist of two or more steps separated by multiple free energy barriers. Although non-classical nucleation theories may be a more sustainable model, the atomistic mechanisms and structural evolutions during nucleus formation in non-classical nucleation pathways are not well known; and remains a challenge for experimental techniques to unravel.
Now, for the first time, a group of researchers at HKUST led by Prof. Xuhui Huang from the Department of Chemistry combined Markov State Models (MSMs) – which model long-timescale dynamics of chemical molecules - and transition path theory (TPT) – which describes the reaction pathway of rare events - to elucidate the ensemble pathways of HIN. MSMs identify intermediate states of disordered ice mixtures and compare parallel pathways (classical vs. non-classical). This advantage helped unravel the underlying mechanisms of non-classical nucleation processes and the co-existence of the two pathways.
These researchers show that the disordered mixing of ice stabilises the critical nucleus and makes the non-classical nucleation pathway as accessible as the classical pathway, whose critical nucleus mainly consists of potential energy-favoured ice. They also discovered that at elevated temperatures, the nucleation process prefers to proceed via the classical pathway since the potential energy contributions, which favour the classical pathway, prevail.
“Not only does our work uncover the mechanisms of non-classical nucleation processes, but it also demonstrates how the combination of MSMs and TPT offers a powerful framework to study structural evolutions of ice nucleation processes,” said Prof. Huang. “More importantly, this method can be extended to other crystal nucleation processes that are challenging to study, which will open new doors for scientists attempting to predict and control crystallization processes.”
The findings were recently published in the scientific journal Nature Communications. The first author of this work: Dr. Chu Li is a long-time HKUST affiliate who completed his PhD, and currently conducts his post-doctoral training at HKUST.
An international research team led by scientists from the Hong Kong University of Science and Technology (HKUST) has developed a novel strategy using brain-wide genome-editing technology that can reduce Alzheimer’s disease (AD) pathologies in genetically modified AD mouse models. This advanced technology offers immense potential to be translated as a novel long-acting therapeutic treatment for AD patients.
In China alone, over 500,000 patients are estimated to be living with a hereditary form of AD - familial Alzheimer’s disease (FAD), which is a congenital form of AD highly associated with family history. Although FAD has a clear genetic cause and can be diagnosed before cognitive problems occur, no effective treatment currently exists.
There is enormous potential in the use of genome-editing technology1 as therapeutic strategies for diseases caused by inherited mutations, such as FAD. It is especially useful for correcting disease-causing genetic mutations before symptoms appear, for which it is considered a “once-and-for-all” treatment as its effects can last a lifetime. However, several hurdles have prevented its clinical development and application - most notably the lack of an effective, efficient, and non-invasive means to deliver genome-editing agents into the brain. Furthermore, existing genome-editing technologies are unable to generate beneficial outcomes throughout the whole brain.
Recently, a team led by Prof. Nancy Ip, Vice-President for Research and Development at HKUST, developed a new genome-editing system that not only crosses the blood–brain barrier, but also delivers an optimized genome-editing tool to the entire brain. Using a newly engineered delivery vehicle for genome-editing, this strategy achieves efficient brain-wide genome editing through a single non-invasive intravenous administration. This effectively disrupts FAD-inflicted mutations in AD mouse models and ameliorates AD pathologies throughout the entire brain, paving the way to novel therapeutic development for the disease.
Meanwhile, the research team also found in the mouse models that the level of amyloid, a protein thought to drive neurodegeneration in AD, remained low for 6 months post-treatment (about 1/3 of their normal lifespan), demonstrating that this single-shot genome-editing strategy has lasting effects. More importantly, no side effects were detected so far in the mice.
“As the first demonstration of efficient brain-wide genome editing to alleviate Alzheimer’s disease pathology throughout the whole brain, this is really an exciting development,” said Prof. Ip, who is also the Morningside Professor of Life Science and Director of the State Key Laboratory of Molecular Neuroscience at HKUST. “Our work is an important milestone for the use of genome editing in treating hereditary brain diseases, and contributes to the development of precision medicine for inherited forms of neurodegenerative diseases.”
Genome editing of the familial mutation in AD mice improves their memory performance.
This research was a collaborative effort among scientists from HKUST; the California Institute of Technology; and the Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences. The results were recently published in Nature Biomedical Engineering.
1Genome editing is a technology that precisely modifies a living organism’s genomic DNA by deleting, inserting, or replacing the DNA at specific locations of the genome.
Sixty years ago, the whole world was gladly amazed when the first human blasted into space; over the years, hundreds of exploration missions have been deployed to extend our knowledge and understanding of the universe. Of all the space mysteries scientists still hope to solve, black holes remain the strangest and most mysterious phenomena we know so little about.
After decades of effort, scientists finally reached a major milestone when they came face to face with a black hole by capturing its first-ever image in 2019, and a more detailed one earlier this year. The ground-breaking images show the supermassive black hole at the center of Messier 87, a galaxy about 54 million light-years away from Earth. The black hole's mass is equivalent to 6.5 billion suns.
Einstein’s theory wins again
In fact, a few years ago, “if you asked astronomers if there could be a black hole 100 times as heavy as the sun, they would have told you that did not exist,” says WANG Yi, Associate Professor at the Department of Physics. “But now, we’ve discovered them!”
The image of the mysterious structure has provided insight into physics and let us understand more about black holes in many ways. “While there have been alternative theories to describe the strong gravity near the black hole horizons, this photo shows that Einstein’s general relativity wins again. What we see in the picture, everything looks like what Einstein predicted, 100 years ago,” he says. The shape of the black hole’s shadow reaffirms Einstein’s theory.
The image also offers insight into the formation and behavior of black hole structures, such as the accretion disk that feeds matter into the black hole and plasma jets that flow from its center. “Because of the image, we now have a better understanding of the physics of the jet,” he says.
Another “eye” opened
Scientists are also studying gravitational waves, the ripples in space that happen when two bodies, such as planets or stars, orbit each other, as Einstein predicted. The first such event was observed in 2015 when two black holes merged. “It’s a new, independent channel to see the universe. Previously, we were only studying electromagnetic waves which is light. It opened a new era of gravitational wave astronomy,” he says.
“It’s like we previously looked at the universe with just one eye, and now with gravitational waves, we have another eye opened!”
For a very long time, it was thought that capturing an image of a black hole was impossible. The resolution needed is like an astronaut standing on the moon seeing what’s in your plate for dinner on the earth.
“To see distant objects, you need a telescope with high resolution. The larger the lens, or aperture, of the telescope, the greater its ability to gather light, and the higher its resolution. In this case, we needed a lens as big as the earth,” he says.
This was possible thanks to a technology that let different telescopes across the globe work together over years, effectively creating a telescope the size of earth.
Why, you may ask, is it important to research something so far from earth? The image has helped scientists, including Prof. Wang, gain knowledge beyond the study of black holes. Ultimately, they have shed light on how a galaxy is formed, how supernovae explode, and, fundamentally, where we come from, he says.
The value of fundamental physics
In addition, knowing about these celestial bodies is a very strong driver for technological development, says Prof. Wang. To explore the unknown of science – be it something as big as the universe or as small as fundamental particles – we need cutting-edge technology to help, and so the pursuit of fundamental science encourages technological development.
An expert in theoretical high energy physics and cosmology, Prof. Wang published over 90 research papers since 2007 focusing on fundamental physics. Recently, he is working on black holes companioned by pulsars, a type of rotation neutron star that helps inform extreme states of matter. He also works on the early universe, examining questions like what happened right after the big bang? What is the fate of the universe?
But the value of fundamental science is especially evident when you take the long view. “Quantum mechanics was discovered about 100 years ago. At the time, what’s the application? Nobody knew. But now, computer technology relies heavily on the theory of semiconductors, a consequence of quantum mechanics, in particular energy band of condense matter,” he says.
“To see the real impact of our research, we may need to wait for 100 years or more,” he says. “But before waiting for 100 years, now we also appreciate the knowledge about how things work in the most fundamental way. It is important to know, even if we don’t know its applications.”
Curiosity drives progress
Prof. Wang’s curiosity rubs off on many students who have taken his course Modern Physics.
“I really hope that all my students are curious about science, not just about exams and coursework. I want them to be driven by curiosity and willing to find out how things work before going to bed every day,” says Prof. Wang.
Shedding Light on Nutritional Complementarity and Biomaterial Production
Researchers from The Hong Kong University of Science and Technology (HKUST) decoded for the first time the chromosomal-level genome of a deep-sea gutless tubeworm and how the worm’s co-living bacterial partners manufacture organic nutrients for its host so it can survive the extreme habitat. The discovery lays foundation for potential applications such as biomaterial production and microbial growth control.
Living in deep-sea hydrothermal vents and cold seeps ecosystems characterized by darkness, high pressure and often high concentrations of toxic substances, submarine tubeworms - common living organisms of such extreme environments, were known to owe their survival and fast growth to sulphide-oxidising symbiotic bacteria that live inside their body. However, the success behind such a complementary “marriage” between the tubeworms and their co-living bacteria had remained unknown due to the lack of genomic resources.
Now, a research team led by Prof. QIAN Peiyuan, Head and Chair Professor of HKUST’s Department of Ocean Science as well as Prof. QIU Jianwen, Professor of Hong Kong Baptist University’s Department of Biology, managed to sequence and assemble the genomes of both the tubeworm and its symbiotic bacteria, which the team collected at around 1,400 meters below sea level from a cold seep in the South China Sea.
Using integrated genomic, transcriptomic and proteomic analyses, the team discovered that the bacteria is highly versatile and can use thiosulfate, carbon monoxide and hydrogen as alternative energy sources. “The bacterium possesses complete metabolic pathways which can generate energy from the toxic substance hydrogen sulfide, and turn simple inorganic molecules such as carbon dioxide into nutrients such as carbohydrates, amino acids and vitamins/cofactors,” said Prof. Qian, who is also HKUST’s David von Hansemann Professor of Science. In addition, the tubeworm endosymbiont has evolved elaborate strategies to distract the host’s protective immunity and evade its defenses. Meanwhile, the worm’s genome has also been remodeled to facilitate symbiosis by not only encoding enzymes to digest the symbionts for nutrients, but also containing a programmed cell death pathway to maintain the bacterial population to a level that is most optimal to the worm itself.
To understand the mechanisms of the formation of tubeworms tube – a unique supporting structure for them to acquire inorganic matter from the seabed, the team further analyzed the proteins of the tubeworms’ tough chitinous tube. They discovered 35 chitin tube matrix proteins, including synthases that manufacture chitin microfibrils and secrete them to the extracellular matrix, chitin-binding proteins which cleave chitin and provide a polymer framework for the organic matrix, and proteins that enhance the toughness of the tube and remodel the chitin scaffold.
“Our finding opens new directions for potential applications – such as using the unique enzymes of the biosynthesis process for biomaterial production; developing strategies to remediate nutritional deficiency, or designing methods on microbial growth control,” added Prof. Qian.
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An international research team led by HKUST has developed a simple but robust blood test from Chinese patient data for early detection and screening of Alzheimer’s disease (AD) for the first time, with an accuracy level of over 96%.
Currently, doctors mainly rely on cognitive tests to diagnose a person with AD. Besides clinical assessment, brain imaging and lumbar puncture are the two most commonly used medical procedures to detect changes in the brain caused by AD. However, these methods are expensive, invasive, and frequently unavailable in many countries.
Now, a team led by Prof. Nancy IP, Vice-President for Research and Development at HKUST, has identified 19 out of the 429 plasma proteins associated with AD to form a biomarker panel representative of an “AD signature” in the blood. Based on this panel, the team has developed a scoring system that distinguishes AD patients from healthy people with more than 96% accuracy. This system can also differentiate among the early, intermediate, and late stages of AD, and can be used to monitor the progression of the disease over time. These exciting findings have led to the development of a high-performance, blood-based test for AD, and may also pave the way to novel therapeutic treatments for the disease.
“With the advancement of ultrasensitive blood-based protein detection technology, we have developed a simple, noninvasive, and accurate diagnostic solution for AD, which will greatly facilitate population-scale screening and staging of the disease,” said Prof. Nancy Ip, Morningside Professor of Life Science and the Director of the State Key Laboratory of Molecular Neuroscience at HKUST.
The work was conducted in collaboration with researchers at University College London and clinicians in local hospitals including the Prince of Wales Hospital and Queen Elizabeth Hospital. The discovery was made using the proximity extension assay (PEA) - a cutting-edge ultrasensitive and high-throughput protein measurement technology, to examine the levels of over 1,000 proteins in the plasma of AD patients in Hong Kong.
As the most comprehensive study of blood proteins in AD patients to date, the work has recently been published in Alzheimer’s & Dementia: The Journal of the Alzheimer’s Association, and has also been featured and actively discussed on different scholarly exchange platforms on AD research such as Alzforum.
AD, which affects over 50 million people worldwide, involves the dysfunction and loss of brain cells. Its symptoms include progressive memory loss as well as impaired movement, reasoning, and judgment. While patients often only seek medical attention and are diagnosed when they have memory problems, AD affects the brain at least 10-20 years before symptoms appear.
Researchers from the Hong Kong University of Science and Technology (HKUST) and Beijing Tiantan Hospital have recently uncovered a new gene mutation responsible for the non-familial patients of cerebral cavernous malformation (CCM) - a brain vascular disorder which inflicted about 10~30 million people in the world.
While the mutation of three genes: namely CCM1, CCM2, and CCM3, were known to be a cause of CCM – they mostly targeted patients who has family history in this disorder – which only account for about 20 per cent of the total inflicted population. The cause for the remaining 80 per cent non-familial cases, however, were not known.
Now, using next-generation sequencing and computational approach, a research team led by Prof. WANG Jiguang, Assistant Professor from HKUST’s Division of Life Science and Department of Chemical and Biological Engineering, in collaboration with Prof. CAO Yong from the Beijing Tiantan Hospital, analyzed the genomic data of 113 CCM patients and identified another mutation called MAP3K3 c.1323C>G, which is found to be responsible for almost all the tested cases who developed popcorn-like lesions in their brain arteries - the most common one among the four types of CCM lesions1(type II CCM).
At present, magnetic resonance imaging (MRI) is a commonly used non-intrusive means that doctors can base upon for diagnosis and treatment. However, the MRI images can only tell the size and type of the lesions, but not the gene responsible for the problem – which can only be ascertained by surgery and laboratory tests. Now, the HKUST research team designed a computational method that could help assess the probability of connection between the lesion shown in the MRI image to the genetic mutation MAP3K3 c.1323C>G. So CCM patients with this gene mutation may be able to receive more targeted treatment without having to undergo surgery – which could bear serious risks including cerebral hemorrhage or new focal neurological deficits.
Prof. Wang from HKUST said, “Our research opens a new direction to the genetic landscape of CCM and uncovers clues to the correlation between MAP3K3 c.1323C>G gene mutation and type II CCM. The design of the computational method, or decision-tree model takes us a step closer to non-invasive diagnosis of CCM cause, and we hope the discovery could help pave way for candidate drug target and therapy development, bringing benefits to patients in the near future.”
The findings were recently published in The American Journal of Human Genetics.
1Type II CCM was said to be the most common among the four types of CCM: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6924279/
(This news was originally published by the HKUST Public Affairs Office here.)
(This article was published on EurekAlert! on May 4, 2021)
A group of researchers at the Hong Kong University of Science and Technology (HKUST) has uncovered the mechanism of how DNA is being melted to start bacterial gene transcription and how one class of antibiotics inhibits this process – an important way in killing bacteria. This discovery provides useful insight on the development of new antibiotics for bacteria that is antimicrobial resistance.
The emergence and spread of new forms of resistance remains a concern that urgently demand new antibiotics. Transcription is a vital process in bacterial cell, where genetic information in DNA is transcribed to RNA for the translation of proteins that perform cellular function. Hence, transcription serves as a promising target to develop new antibiotics because inhibition the transcription process should effectively kill the bacteria. Bacterial RNA Polymerase, the core enzyme for transcription, must load the DNA and separate the double-stranded DNA to single stranded DNA to read the genetic information to initiate transcription. This process is also called DNA melting and is facilitated by the opening and closing of the loading gate of RNA Polymerase. The loading gate contains two flexible pincers (clamp and β-lobe) resembling the shape of a crab claw. DNA melting via this loading gate is a multi-step and highly dynamic process, and it provides a promising strategy for the design of novel antibiotics by inhibiting this process. Yet, the understanding of DNA melting requires a detailed understanding in the movements and dynamics of the loading gate, the lack thereof hampers future development of antibiotics.
To offer new direction for more effective therapeutics, a research team led by Prof. Xuhui HUANG, Department of Chemistry and Department of Chemical and Biological Engineering at HKUST, recently discovered the working mechanism of an antibiotics, Myxopyronin, by targeting the movement of the loading gate to inhibit the DNA melting prior to bacterial gene transcription. The research team identified a partially closed form of the flexible clamp domain, into which an antibiotic called Myxopyronin can bind with. The binding of Myxopyronin to the RNA Polymerase diminishes the gate's ability to close, eventually inhibiting the DNA melting, which is vital for the survival of the bacteria.
More interestingly, the research team also found the unprecedented role of the β-lobe during the loading of DNA to the inner cleft of RNA Polymerase. They discovered that the opening of β-lobe is sufficient to accommodate the loading of double helix DNA without opening the Clamp. The role of β-lobe has not been previously reported, and this finding opens the opportunity to the development of new antibiotics targeting the β-lobe of RNA Polymerase to halt transcription.
"The shape of bacterial RNA polymerase resembles a crab claw that works like a pincer. The shape and flexibility of the two pincers are important for RNA Polymerase to hold and separate the double-helix form of DNA into single-stranded. We showed that an antibiotic that targets the movement of the pincers would be a promising as a drug candidate" said Prof. Huang. "What is more exciting, is that we also discovered a novel critical role of the β-lobe that can serve as a new target for future antibiotics development."
This work is made possible only with the quasi-Markov State Model (qMSM) recently developed in Prof. Huang's lab. qMSM is built from extensive all-atom molecular dynamics simulations, and successfully predicts dynamics of RNA Polymerase's loading gate at atomic resolution and millisecond (10-3 second) timescale. This new method adopts the generalized master equation formalism to encode non-Markovian dynamics, which has advantages over the popular Markov State Models based on Master Equation. Hence, it is especially promising to be applied to study complex conformational changes of proteins.
The first author of this work: Dr. Ilona UNARTAR is a long-time HKUST affiliate who completed her undergraduate, PhD, and currently conducts her post-doctoral training all at HKUST from Department of Chemistry and Bioengineering graduate program. Other collaborators of this work come from Kyoto University and King Abdullah University of Science and Technology.
This study was recently published in the scientific journal Proceedings of the National Academy of Sciences.
(This article was published on EurekAlert! on April 28, 2021)
It is challenging to analyze proteins at low concentrations, especially for those in a mixture of various conformations such as intrinsically disordered proteins (IDPs). A research team led by Prof. HUANG Jinqing, Assistant Professor of Department of Chemistry at The Hong Kong University of Science and Technology (HKUST), has developed optical tweezers-coupled Raman spectroscopy that can directly probe the structural features of alpha-synuclein, an IDP closely linked to Parkinson’s disease, at the physiological concentration by focusing on individual protein molecules.
IDPs play an important role in biological processes and many of them are associated with incurable neurodegenerative diseases. As a typical IDP, alpha-synuclein lacks a stable 3-D architecture known as secondary structures. It spontaneously undergoes conversions from one secondary structure to another, which could eventually result in the buildup of protein aggregates involved in Parkinson’s disease pathology. However, the transient species during the conversion possess various structures and exist in low population among a dynamic equilibrium mixture. Therefore, their structural features are usually buried under the detection results from traditional measurement techniques, which average the signals detected from large sample quantities and long detection time.
In the study, Prof. Huang and her collaborators integrate optical tweezers and surface-enhanced Raman spectroscopy (SERS) in a novel platform to generate tunable and reproducible SERS enhancements with single-molecule level sensitivity in aqueous environments, in order to characterize these IDPs while maintaining their intrinsic heterogeneity with great biological significance. Specifically, a hotspot can be visualized and controlled by optical tweezers to allow proteins to go through in a microfluidic flow chamber, which makes it convenient to adjust the measurement parameters in the real time for the in situ spectroscopic characterizations. It directly identifies the structural features of the transient species of alpha-synuclein among its predominant monomers at physiological concentration of 1 μM by reducing the ensemble averaging in quantity and in time, providing profound insight to understand the initiation of amyloid protein aggregation. Hence, this SERS platform has great potential to reveal the structural information of IDPs in the dynamic, heterogeneous, and complex biological systems.
“Our strategy enables the precise control of the hotspot between two trapped micrometer-size silver nanoparticle-coated silica beads to improve the SERS efficiency and reproducibility in aqueous detections. Except for the tunable SERS enhancement, the integrated optical tweezers also offer sub-nanometer spatial resolution and sub-piconewton force sensitivity to monitor light-matter interactions in the plasmonic hotspot for extra physical insight. More importantly, our method opens a new door to characterize the transient species of IDPs in dilute solutions, which remains a significant challenge in the biophysics community. Ultimately, it will be exciting to fully exploit the precise force manipulation of the integrated optical tweezers to unfold a single protein inside the controllable hotspot and resolve its structural dynamics from the endogenous molecular vibrations by the integrated Raman spectroscopy.” said, Prof. Huang.
The study was recently published in the scientific journal Nature Communications.