New research from the Kindt Group was recently featured in eScienceCommons. The Kindt Group, in collaboration with students in the Mathematics and Computer Science Department, has developed a new method to calculate equilibrium constants using small-scale simulations. The software, which reduces computing time using tricks derived from number theory, has been named PEACH for “partition-enabled analysis of cluster histograms”. Moving forward, this method will give scientists the ability to simulate the behavior of numerous molecules and explore how molecular structures dictate assembly.
“‘Our method will allow computational chemists to make better predictions in simulations for a wide range of complex reactions — from how aerosols form in the atmosphere to how proteins come together to form amyloid filaments implicated in Alzheimer’s disease,’ says James Kindt.”
Sometimes, being in an academic lab setting can feel a bit pointless. Instructors and TAs are there to help you every step of the way, procedures are laid out for you step-by-step, and everyone pretty much knows what the “right” result should be. I understand that this method helps you learn techniques and reinforce concepts, but it definitely isn’t what I’ve experienced in a real research setting.
Dr. Jeremy Weaver’s analytical chemistry lab has been a fun and fulfilling change of scenery from step-by-step lab work. Our class visited the WaterHub with sample collection bottles and got a hands-on look at the real science that goes on there (I talk more about the WaterHub experience here). Then, we took the samples back into the lab to do some real research.
Dr. Weaver famously says that analytical chemistry is the class where data accuracy and precision matter the most. But for the WaterHub project, he took a more open-ended approach. He didn’t give us a procedure to follow; instead, we spent a week scouring the Internet and the scientific literature to figure out what to do. And when we asked if a certain procedure would work, Dr. Weaver encouraged us to go for it, give it a shot, and see what happened.
Using the techniques we learned in lab, including gas chromatography, titrations, and spectrophotometry, we determined (somewhat successfully) the phosphate and aluminum concentrations of the water, along with “water hardness” – a fancy term for the concentrations of calcium, magnesium, and a few other ions in a water sample. These are values that water quality testers would measure during a routine check of water quality.
Of course, without a surefire procedure to follow, it took a couple of tries to work out the kinks. My portion of the project was to determine the phosphate concentration of the WaterHub samples using UV/Vis spectrometry. The concept behind this technique is simple – you add an agent to your sample that creates a color change, and the degree to which the color appears corresponds to the concentration of the sample. The first time I added my coloring agent to each sample, absolutely nothing happened – even when I knew that there was a ton of phosphate in the sample!
The process of research, as we learned, is full of troubleshooting and setbacks. But eventually, I found the amount of phosphate in the WaterHub water! Boy, did I feel accomplished because I found the procedure and performed the experiments myself. Even in an academic lab setting, it is possible to conduct real research, answer real questions, and engage with the Emory community on a larger level. Dr. Weaver’s WaterHub project brought the esoteric techniques of quantitative analytical chemistry and gave them new life through a real-life application.
Laura Briggs is a sophomore majoring in chemistry and dance. Laura is a Woodruff Scholar and the Vice President of the Emory Swing Dance Club. She is also a member of the Emory Dance Company and hosts a weekly, science-themed radio show. Laura is a research assistant in the Weinert lab, where she studies really cool bacteria that attack potatoes. Laura plans to pursue either a Ph.D. in biochemistry or a master’s in science writing.
To learn more about the WaterHub, check out this link from Campus Services!
Yang Liu (Salaita Group) is bringing new techniques to the emerging field of mechanobiology; at the same time, he’s returning to his roots.
Literally. As in, plants.
Yang’s father is an academic biologist studying agriculture in China.
“I think in the beginning, my dad really wanted me to be a biologist,” says Yang. “But normally kids don’t want to pursue the same career path as their parents.”
As an undergraduate in China, Yang started out studying mechanical engineering. Then, he attended a general chemistry lecture with a famous chemistry professor who made a convincing case for the importance of the discipline. “He said, ‘chemistry is the central science connecting physical sciences, life sciences and applied sciences all together,’’ says Yang. “And I was so fascinated by it. And I changed my major.”
At Emory, Yang joined the lab of Khalid Salaita. His research in the Salaita Group takes a novel approach to a common scientific question: how does the immune system recognizes and eliminates “invaders”, such as pathogens or cancer cells? Most research explores how chemical signals mediate this process. Yang’s work expands on existing work in the Salaita Group that focuses on mechanical signaling—the way that immune cells physically probe their targets within the body. “Cells can touch and apply forces to one another,” explains Yang, a process he refers to as a “handshake.” Yang’s research develops tools that allow scientists to “see” these kinds of physical interactions.
Specifically, Yang has developed a technique named molecular tension fluorescence microscopy (MTFM) that employs single elastic molecules—DNA, protein, and polymer— as sensors to visualize membrane receptor mediated forces at the piconewton level. “One piconewton is the weight of one trillionth of an apple and surprisingly, pN forces regulate biochemical signaling pathways,” says Yang. These forces are too small for scientists to measure using conventional methods. Existing tools aren’t sensitive enough or they are inefficient.
“Until our method kicks in,” says Yang.
Yang has combined nanotechnology and the “easy” surface chemistry of gold nanoparticles to make MTFM probes more effective. “These gold particle sensors are spring scales at nanoscale ,” says Yang. “Compared to previous techniques, these probes are of significantly enhanced sensitivity, stability and amenable for detecting forces mediated by almost all kinds of cell receptors.”
The improvements have caught the attention of researchers in other Emory units—and even nationally and internationally. Yang has collaborated with the Evavold Lab in the Department of Immunology at Emory to help them measure mechanical forces mediated by different immune cells. He also has collaborators from as far away as New York and Germany.
Regarding these collaborations, Yang says: “The need to be trained [to use this method] is very high. The method is not hard, it’s easy. So people usually spend a few days and they should be able to master it…and we still maintain quite tight collaboration. We not only teach them how to make it, we actually get involved in the scientific questions they care about and continue this collaboration.”
Recently, Yang’s success in developing the new method was recognized with the department’s highest graduate student honor, the Quayle Outstanding Student Award. Speaking of Yang’s progress shortly after the award ceremony, advisor Khalid Salaita praised Yang’s work ethic as well as his science: “Yang was a real pleasure to have in the lab. He was incredibly thoughtful, well read, and intensely motivated. More than anyone else I’ve worked with, Yang displayed a keen instinct for experimental design. He spent countless hours in the dark microscope room collecting data and working around the clock fueled up with his favorite bbq Pringles and excited by the science.”
The award ceremony was followed swiftly by another milestone—a successful PhD defense. Next, Yang is headed to John’s Hopkins University where he will work in the lab of Dr. Taekjip Ha, a world leader in the development of single molecule fluorescence microscopy and force spectroscopy.
Yang’s pioneering research wasn’t always smooth sailing. “I didn’t get my first experiment done until the first semester of my third year. Everything before that didn’t work.” He credits his perseverance to his father’s example—“agriculture is even slower, waiting for the growth of plants. You can only do two experiments a year!”—as well as his own scientific curiosity. His advisor, Khalid Salaita, was also an inspiration throughout the process. “He is always passionate and ignited my love for science. You love it and you work hard to make something meaningful to the society and also make yourself valuable, so, that’s what I’d like to do and that’s because of these two people.”
Does all this mean that Yang has overcome his initial reluctance to follow in his father’s footsteps towards biology?
“I think I’m going back to the route, mining chemistry, biology. In the beginning I was against it, but I do like it.” Still, chemistry has his heart. “Chemists not only create new tools, new theories and new materials, but also create new opportunities. And if you want to study biology as a chemist, there are some advantages too because you can understand and explore the secret of life at the molecular level.”
The hallmark of Alzheimer’s disease is the presence of plaques in the brain formed by the aggregation of Aβ peptide with heavy β-sheet content–also known as amyloid. Amyloid is hypothesized to be causative in Alzheimer’s disease through multiple mechanisms such as oxidative stress, interaction with receptors and synaptic loss. Currently, over five million Americans are living with Alzheimer’s disease, costing the nation 236 billion a year. It’s expected that by 2050,healthcare spending on Alzheimer’s will reach one trillion. The NIH invests around 500 million annually for Alzheimer’s research. Despite the prevalence of Alzheimer’s and the intensive efforts of researchers, no effective therapeutics for the disease is yet available. This dilemma attracted me to the study of amyloid as my PhD research project.
Current drug design for Alzheimer’s disease focuses on finding molecules that bind and block the action of these deleterious proteins. Typically, a disease—like cancer, diabetes, and, as some have believed, Alzheimer’s—is caused by proteins with a fixed structure. However, my study in Dr. David Lynn’s lab at Emory University demonstrates that amyloid, unlike conventional drug targets, is highly dynamic and can change structure over time. My research could potentially explain why conventional drug discovery methods don’t succeed with Alzheimer’s –they generally ignore the structural diversity and the changing nature of amyloid.
The peptide I use in this research is the nucleating core of Aβ Dutch mutant, Aβ(16-22)E22Q or KLVFFAQ. People with this genetic mutation develop a more severe form of Alzheimer’s. I discovered that early on, after dissolving, this peptide forms ribbon shaped structures and later autocatalytically change into fibers (Figure 1). More detailed characterization using IE-IR (isotope edited infrared spectroscopy) and solid state NMR (Nuclear Magnetic Resonance) reveals that in the ribbon shape, two neighboring peptides within a β-sheet are pointing in the opposite direction—a state that is commonly referred to as an anti-parallel β-sheet arrangement. Yet the conformation is transient. After a week, the peptides autocatalytically switch into parallel β-sheet where all peptides are pointing in the same direction. Furthermore, by simply adding salt, I was able to control the speed of such shape shifting and even greatly expand the range of observed structures.
This research is significant in the study of Alzheimer’s disease and drug development because it begins to explain why no effective therapeutics have been developed for Alzheimer’s disease. Due to the high thermo-stability of amyloid, researchers commonly assume amyloid structure remains static upon assembly. My study demonstrates the opposite: amyloid can change structure and such a change is sensitive to environmental conditions. Now people can imagine the change and diversity that could occur when amyloid is spreading through different cellular environments as it ravages the brain.
Such an “environmental dependent conformational change” is an important property of Aβ protein and these dynamics are beginning to gain more attention in the scientific community despite being counter-intuitive. Amyloid’s high thermostability has led researchers to reason that once formed amyloid should be stable and their structure should be faithfully replicated throughout the brain. The implication of my study on the treatment of Alzheimer is that instead of measuring the amount of amyloid and treating patients non-discriminately, the structure diversity of amyloids should be central to any consideration in developing diagnostics and therapeutics. New methods of drug discovery—taking into account amyloid’s unique properties—will certainly be necessary for treating Alzheimer’s and the increasing number of amyloid diseases.
Enzymes are responsible for catalyzing a myriad of reactions necessary for life. Because enzymes play such an important role in human physiology, they are often targets for drugs and disease treatments. Naturally occurring enzymes are capable of catalyzing a wide variety of reactions, but imagine if we could design an enzyme to catalyze any reaction we wanted. We would be able to create new antibiotics easily to combat antibiotic resistance or to quickly synthesize chemicals for industrial applications. Scientists have made a lot of progress towards creating new enzymes, yet there are still roadblocks. Modifying existing enzymes through directed evolution is inefficient and limited by the need for high throughput screening methods. Conversely, in the case of rational design, we are missing key information for the technique to work at its full potential.
My research works to fill in the gaps in our knowledge to allow for the efficient development of new enzymes. A large portion of the scientific community focuses on determining the structure of enzymes and how the structure impacts function. While this work is enormously important, it doesn’t tell the full story. One major aspect that is often overlooked when examining structure-function relationships is that enzymes are dynamic molecules. This means that they physically move, bend, wiggle, and change shape during catalysis.
To study enzyme dynamics, I use temperature jump spectroscopy. There are only a few labs around the world that use this technique, and even fewer that use it to study enzymes. Temperature jump spectroscopy relies on rapidly initiating a change in equilibrium. For example, my samples contain enzymes and ligands. As determined by the equilibrium constant, some of the ligand is bound and some ligand is free in solution. The sample starts at equilibrium at a specified temperature. Then, a laser pulse is used to rapidly heat a small portion of the sample. The system must relax to a new equilibrium at the higher temperature. Since ligand binding is an exothermic reaction, there will be a net flux of ligands dissociating from the enzyme. However, as a system relaxes to a new equilibrium it will shift in the forward and reverse directions providing information about both processes. From this data I can determine the rate at which ligands are binding and unbinding, accompanying enzyme motions, and even conformational changes unrelated to ligand association. These changes occur on the microsecond timescale.
Although temperature jump spectroscopy could be applied to any number of enzymes, so far I’ve studied one enzyme in particular, dihydrofolate reductase (DHFR). It is a small ubiquitous enzyme that is well known for changing conformations during its catalytic cycle. Thus, it is a good starting place for understanding enzyme dynamics. Furthermore, DHFR is an important enzyme for nucleic acid synthesis. Since nucleic acid synthesis is necessary for cellular replication, DHFR inhibition is a strategy for anticancer and antibacterial agents.
Understanding the motions of DHFR could lead to the development of new inhibitors to combat resistance developed in certain cancers. The technique I use can be applied to other enzyme systems as well. By studying multiple enzymes we can build an understanding of enzyme motions in general, which can then be used to inform computational simulations for rational enzyme design. This would ultimately allow us to efficiently design new enzymes as well as new drugs.