Meredith Brenner
Research Summary
While supported by the Molecular Biophysics Training Grant, Meredith Brenner began developing a new multiphoton Förster Resonance Energy Transfer, or FRET, microscopy technique using ultrafast laser pulse shaping.
This technique is designed to selectively excite multiple fluorophores in biological systems. It can also provide quantitative information about donor and acceptor molecule concentrations and their changes over time during cellular processes.
Training Grant Research
During her time on the training grant, Meredith implemented ultrafast pulse shaping and used it to selectively excite fluorophores in solution.
She later applied this pulse-shaping technique to live-cell imaging. Using live Cos-7 cells expressing fluorescent proteins, she obtained fluorescence images, achieved selective excitation of fluorophores in cells, and observed FRET in live cells.
She also learned to perform fluorescence lifetime measurements and used those results to determine the FRET efficiency of her system.
Technical Skills and Analysis
Meredith developed MATLAB code to:
- Design pulse shapes
- Process and analyze images
- Support quantitative analysis of FRET results
She also extended the existing FRET theory to help quantify her experimental results.
Meredith is currently performing calculations on her live-cell image data. She is also exploring other applications of ultrafast pulse shaping for live-cell imaging, including:
- Selective excitation in different biological systems
- Imaging systems that contain multiple spectrally overlapping fluorophores
Research Training
Meredith has received training in:
- Ultrafast laser use
- Basic wet lab techniques
- Two-photon laser scanning microscopy
- Tissue culture techniques
- Cell maintenance, transfection, and live-cell imaging
She has also completed coursework on nonlinear optical and biophysical techniques, including their theoretical foundations and recent advances in the field.
Publications
“Pulse-shaping-based Two-photon FRET Microscopy,” Meredith H. Brenner, Dawen Cai, Samuel W. Straight, Joel A. Swanson, and Jennifer P. Ogilvie. Proceedings of Ultrafast Phenomena 2012.
“Pulse Shaping Multiphoton FRET Microscopy.” Meredith H. Brenner, Dawen Cai, Sarah R. Nichols, Samuel W. Straight, Adam D. Hoppe, Joel A. Swanson, and Jennifer P. Ogilvie. Proceedings of SPIE 8226, 82260R, 2012.
Taeyjuana Curry
Taeyjuana Curry began her research in the Kopelman Lab by refining techniques for the external calibration of organically modified silane, or ORMOSIL, nanoparticles. These nanoparticles were used for intracellular electric field sensing in cancer cells.
In recent years, her research has focused primarily on nanoparticle-mediated photothermal therapy, or PTT, for cancer cells.
Her work has shown that covalently linked Coomassie Blue dye polyacrylamide, or PAA, nanoparticles can be used for effective photothermal therapy of cancer cells. This research supports a multimodal approach to cancer detection and treatment using the same nanoplatform previously used for optical and photoacoustic imaging.
Her most recent research focuses on the efficient delivery of femtosecond laser ablation-generated gold nanoparticles into cancer cell nuclei. This work aims to enable more effective photothermal therapy, as well as other therapeutic approaches.
After completing her Ph.D. program in Physics, Taeyjuana plans to expand her research to include the use of nanoplatforms in the detection, imaging, and treatment of malignant cell lines.
Publications
Antonuk LE, El-Mohri Y, Du H, Behravan M, Curry T, Zhao Q, Koniczek M, Street R, and Lu JP: Exploration of the Potential Performance of Polycrystalline Silicon-based Active Matrix Flat Panel Imagers Incorporating Active Pixel Sensor Architectures. SPIE Medical Imaging 2008 - Physics of Medical Imaging, San Diego, CA, February 16-21, 2008. (Abstract and oral presentation.)
Behravan M, Antonuk LE, El-Mohri Y, Zhao Q, Yeakey M, Martelli C, McDonald J, Curry T and Koniczek M: Noise Characterization of Polycrystalline Silicon Thin Film Transistors for X-ray Imagers Based on Active Pixel Sensor Architectures. Materials Research Society Spring 2008 Meeting: Symposium A – Amorphous and Polycrystalline Thin-Film Silicon Science and Technology, San Francisco, CA, March 24 - 28, 2008. (Abstract and oral presentation.)
Taeyjuana Curry, Tamir Epstein, Ron Smith, & Raoul Kopelman. Photothermal Therapy of Cancer Cells mediated by Blue Hydrogel Nanoparticles. Under review.
Taeyjuana Curry, Wei Qian, & Raoul Kopelman. Surface Optimization of Gold Nanoparticles for Controlled Delivery Into Cancer Cell Nuclei. Submitted.
Josef Dunbar
I am a biochemist with a strong interest in understanding biological systems at the molecular and atomic levels.
As a graduate student in the Biophysics program, I benefit from the program’s interdisciplinary approach. This environment allows me to explore many aspects of protein behavior through the perspectives of chemistry, physics, and biology.
In Dr. Kevin Kubarych’s lab, my research has focused on:
- Developing and programming new instrumentation for acquiring two-dimensional infrared, or 2D-IR, spectra
- Developing new multidimensional spectroscopy methods
- Synthesizing new molecular probes for 2D-IR spectroscopy of biological samples
These projects offer new ways to understand biological processes at the molecular level.
Robert Fick
Research Summary
Robert Fick studies the mechanism of methylation by S-adenosyl methionine, or AdoMet, methyltransferases.
AdoMet is the most common source of methyl groups used to modify proteins, carbohydrates, and nucleic acids. AdoMet-dependent enzymes can catalyze methyl transfer to carbon, oxygen, nitrogen, and sulfur atoms.
Research Background
Previous work in the Trievel Laboratory showed that carbon-hydrogen–oxygen, or CH–O, hydrogen bonds occur between the AdoMet methyl group and two parts of the SET7/9 active site:
- A backbone carbonyl group
- A tyrosine hydroxyl group
These hydrogen bonds help align the methyl group and may facilitate methyl transfer.
Current Research
Robert’s research focuses on other classes of AdoMet methyltransferases. His goal is to determine whether CH–O hydrogen bonding is a common mechanism for methyl transfer across different enzyme classes.
This hydrogen bonding may help stabilize the carbocation-like transition state that occurs during methyl transfer.
Research Methods
Robert uses several biophysical techniques to characterize CH–O hydrogen bonding, including:
- Computational modeling
- Nuclear magnetic resonance, or NMR, spectroscopy
- Infrared spectroscopy
- Site-directed mutagenesis
- Isothermal titration calorimetry, or ITC
- Enzyme kinetic analysis
- Neutron crystallography
- X-ray crystallography
Computational modeling and NMR spectroscopy are used together to study methyl hydrogen chemical shifts. These shifts provide information about the electronic environment of the ligand when it is bound to the enzyme.
Infrared spectroscopy is used to directly observe bond behavior. This work uses perdeutero-methyl AdoMet, in which the methyl hydrogens are replaced with deuterium. The resulting carbon-deuterium stretch is unique to the ligand and can be measured spectroscopically.
Robert also uses point mutations of tyrosines in the enzyme active site to study specific bonding interactions. These mutations include the incorporation of para-aminophenylalanine or phenylalanine.
ITC provides binding data that can be used to compare mutant enzymes with their native forms. These results are combined with kinetic studies to determine the role of specific bonds in enzyme function.
Neutron and X-ray crystallography are used to examine active site structure and determine the positions of methyl hydrogens.
Research Impact
This research may reveal a broader mechanism used by AdoMet-dependent enzymes. It may also identify important molecular contacts that could support future inhibitor design.
Scott Horowitz
At the beginning of Scott Horowitz’s graduate studies, little was known about the function and importance of carbon-hydrogen–oxygen, or CH–O, hydrogen bonding in biology.
To address this gap, Scott initiated a collaboration between the Al-Hashimi and Trievel laboratories. This collaboration brought together multiple biophysical and biochemical techniques and supported the development of new methods to study CH–O hydrogen bonding.
Research Focus
Scott focused his research on the role of CH–O hydrogen bonds in S-adenosyl methionine, or AdoMet, dependent methylation. This process is important for many cellular functions.
Previous studies suggested that the catalytic methyl group might form CH–O hydrogen bonds within the active site of methyltransferase enzymes.
Using multiple techniques, Scott showed that CH–O hydrogen bonds are present in all seven structural classes of AdoMet-dependent methyltransferases. His work also showed that these hydrogen bonds are required for both cofactor binding and catalysis.
Research Findings
Using nuclear magnetic resonance, or NMR, spectroscopy and X-ray crystallography, Scott experimentally determined the atomic-scale structure and motions of the catalytic methyl group and its hydrogen-bonding partners.
His findings showed that these interactions support catalysis in two ways:
- They help stabilize the positively charged transition state.
- They restrict methyl group motion.
These interactions are the only known conserved element across the many structural classes of AdoMet-dependent methyltransferases. For this reason, Scott and his collaborators believe that CH–O hydrogen bonds were a critical part of methyltransferase convergent evolution and are fundamental to the methyl transfer reaction.
Publications
Rafiee P, Theriot ME, Nelson VM, Heidemann J, Kanaa Y, Horowitz SA, Rogaczewski A, Johnson CP, Ali I, Shaker R, Binion DG. Human Esophageal Microvascular Endothelial Cells Respond to Acidic pH Stress by PI3K/AKT and p38 MAPK-regulated Induction of Hsp70 and Hsp27 Am J Physiol Cell Physiol, 291(5) C931-945 (2006)
Horowitz S, Binion DG, Nelson VM, Kanaa Y, Javadi P, Lazarova Z, Andrekopoulos C, Kalyanaraman B, Otterson MF, Rafiee P. Increased Arginase Activity and Endothelial Dysfunction in Human Inflammatory Bowel Disease Am. J. Physiol Gastrointest Liver Physiol, 292(5)G1323-G1336 (2007)
Krishnan S*, Horowitz S*, Trievel RC. Structure and Function of Histone H3 Lysine 9 Methyltransferases and Demethylases Chembiochem, 12(2):254-263 (2011)
Horowitz S, Yesselman JD, Al-Hashimi HM, Trievel RC. Direct Evidence for Methyl Group Coordination by Carbon-Oxygen Hydrogen Bonds in the Lysine Methyltransferase SET7/9, J. Biol. Chem, 286(21):18658-63 (2011)
Horowitz S, Dirk LMA, Yesselman JD, Nimtz J, Del Rizzo PA, Mehl RA, Houtz RL, Al-Hashimi HM, Trievel RC. Methyl CH O Hydrogen Bonds Orchestrate AdoMet-Dependent Methylation Under review
Frank AT, Horowitz S, Andricioaei I, Al-Hashimi HM. Use of 1H NMR Chemical Shifts in Determining RNA Structure and Dynamics In preparation
Horowitz S, Trievel RC. Carbon-Oxygen Hydrogen Bonds in Biology, J. Biol. Chem, invited review article, submitted
Yesselman JD*, Horowitz S*, Trievel RC, Brooks CL. Systematic Analysis of Methyl CH O Hydrogen Bonding in Proteins In preparation
Horowitz S*, Fick RJ*, Trievel RC. Mechanistic Aspects of AdoMet-Dependent Methylation invited review article, to be published May 2013
*denotes equal contribution
Michael Howard
Research Summary
I use crystallography, nuclear magnetic resonance, or NMR, spectroscopy, and enzyme kinetics to study the enzymatic mechanisms and molecular recognition features of plant and human mitochondrial ribonuclease P, or RNase P, enzymes.
Transfer RNAs, or tRNAs, are initially transcribed with extra nucleotides on their 5-prime and 3-prime ends. These extra nucleotides must be removed by tRNA processing enzymes before the tRNAs can function properly.
RNase P is responsible for processing the 5-prime end of precursor tRNAs. This enzyme is conserved across all domains of life.
Traditionally, RNase P enzymes are ribozymes. They use catalytic RNA and magnesium ions to hydrolyze phosphodiester bonds. However, recent research has shown that a protein-only form of RNase P exists in plant and human mitochondria.
Research Progress
I obtained the first high-resolution crystal structure of mitochondrial RNase P and have begun structure-function studies to better understand its enzymatic mechanism.
I have also performed metal-soak experiments using different divalent metals to identify metal-binding sites in the crystal structure.
These metal-soak experiments, combined with metal reconstitution assays, showed that:
- Magnesium, or Mg, activates catalysis
- Manganese, or Mn, activates catalysis
- Calcium, or Ca, does not activate catalysis
- Zinc, or Zn, does not activate catalysis
Substrate Recognition Studies
I am also performing binding and cleavage assays with different precursor tRNA substrates. These experiments are designed to identify regions that contribute to substrate specificity.
Preliminary binding assay data revealed a tightly binding tRNA substrate. This substrate has been used in co-crystal screening to help determine the structure of a protein-tRNA complex.
Preliminary crystals have grown and will soon be analyzed at the Advanced Photon Source at Argonne National Laboratory.
Research Training
I have received training in:
- Protein crystal data collection and analysis
- Fluorescent labeling techniques for tRNAs
- Transient kinetics
- Steady-state kinetics
My coursework has introduced me to these techniques, their theoretical foundations, and recent advances in molecular biophysics. This training has significantly supported my thesis research.
Alex Johnson-Buck
Research Summary
Over the past decade, researchers have become increasingly interested in building nanoscale materials and devices from deoxyribonucleic acid, or DNA.
DNA is useful for nanoscale construction because it can self-assemble in predictable ways. It can also be modified to position and interact with other functional materials.
As the field grows, researchers need a detailed understanding of how design choices affect assembly yield and performance. To address this need, Alex Johnson-Buck uses single-particle fluorescence techniques to study the kinetic and chemical properties of DNA nanomaterials.
Research Techniques
Alex has developed two techniques:
- Nanoscale chemical fingerprinting
- Single-origami kinetic assays
Nanoscale Chemical Fingerprinting
Nanoscale chemical fingerprinting combines single-particle localization with the reversible binding of fluorescent probes to specific features on a target structure.
In this work, the target structure is a DNA origami tile with patterned surface features. The technique creates a quantitative nanoscale map of interactions between the DNA structure and components in solution.
Using a two-color fluorescence approach, Alex can:
- Distinguish different patterns of surface features
- Monitor enzymatic modifications of surface patterns
- Identify stable spatial distributions of binding
These stable binding distributions, or “fingerprints,” are unique to each DNA tile. They are not fully predictable from the tile design alone.
This technique makes it possible to characterize subtle but important chemical properties of individual nanostructures with high spatial and temporal detail.
Single-Origami Kinetic Assays
Single-origami kinetic assays monitor the cumulative fluorescence or Förster resonance energy transfer, or FRET, signal from individual nanoscale DNA origami tiles.
These assays track how DNA origami tiles bind fluorescently labeled components from solution.
Using this technique, Alex found that interactions between adjacent binding sites can significantly affect:
- DNA hybridization kinetics
- Overall assembly yield
These findings are important for applications that assemble components on DNA scaffolds with controlled stoichiometry and spacing.
Publications
Johnson-Buck, Alexander E.; Blanco, Mario R.; Walter, Nils G. (2012). Single-molecule fluorescence resonance energy transfer. In Encyclopedia of Biophysics. 1 (G. Roberts, Ed.), Springer, in press.
Blanco, Mario R.; Johnson-Buck, Alexander E.; Walter, Nils G. (2012). Hidden Markov modelling. In Encyclopedia of Biophysics. 1 (G. Roberts, Ed.), Springer, in press.
Michelotti, Nicole; Johnson-Buck, Alexander; Manzo, Anthony J.; Walter, Nils G. (2011) Beyond DNA origami: the unfolding prospects of nucleic acid nanotechnology. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology. DOI: 10.1002/wnan.170.
Marek, Matt S.; Johnson-Buck, Alexander E.; Walter, Nils G. (2011). The shape-shifting quasispecies of RNA: One sequence, many functional folds. Physical Chemistry Chemical Physics. 13, p. 11524-11537.
Johnson-Buck, Alexander E.; McDowell, Sarah E.; Walter, Nils G. (2011). Metal ions: Supporting actors in the playbook of small ribozymes. In Metal Ions Life Sci. 9 (A. Sigel, H. Sigel, R.K.O. Sigel, Eds.), The Royal Society of Chemistry, Cambridge, UK, in press.
Lund, Kyle A.; Manzo, Anthony J.; Dabby, Nadine; Michelotti, Nicole; Johnson-Buck, Alexander; Nangreave, Jeanette; Taylor, Steven; Pei, Renjun; Stojanovic, Milan N.; Walter, Nils G.; Winfree, Erik; Yan, Hao. (2010). Molecular robots guided by prescriptive landscapes. Nature 465, 206-210.
Michelotti, Nicole, de Silva, Chamaree, Johnson-Buck, Alexander E., Manzo, Anthony J., Walter, Nils G. (2010). A bird's eye view: tracking slow nanometer-scale movements of single molecular nano-assemblies. Methods in Enzymology 475, 121-148.
Johnson-Buck, Alex; Kim, Gwangseong; Wang, Shouyan; Hah, Hoe Jin; Kopelman, Raoul. Fabrication, Characterization, and Spectral Properties of Indigo Blue Nanocrystals. (2009). Mol. Cryst. Liq. Cryst. 501, 138-144.
Josh Jasensky
Josh Jasensky uses vibrational spectroscopy to study chemical behavior in biological systems.
Vibrational spectroscopy is useful because it can identify different chemical signatures within a sample. These methods are especially valuable for non-invasive imaging and surface characterization.
Josh’s thesis research focuses on two main areas:
- Non-invasive analysis of human oocytes using coherent anti-Stokes Raman scattering microscopy
- Characterization of antimicrobial peptides on abiotic surfaces using sum frequency generation vibrational spectroscopy
Non-Invasive Imaging of Human Oocytes
The health, growth, and preservation of human oocytes are the focus of many advanced screening methods. These methods are used to evaluate oocyte viability and the likelihood of eventual fertilization.
The most widely used methods for assessing oocyte health rely on morphological scoring. However, these approaches can be prone to error.
Part of Josh’s thesis research focuses on developing coherent anti-Stokes Raman scattering, or CARS, microscopy as a non-invasive analytical tool. This technique may help with:
- Selecting candidates for cryopreservation
- Improving cryoprotectant exposure protocols
- Increasing oocyte cryotolerance
To date, Josh has successfully developed a method to calculate total lipid content in oocytes. This lipid content has been shown to correlate with specific developmental stages in oocyte maturation.
His long-term goal is to treat oocytes using established cryoprotectant exposure protocols and determine whether measurable chemical differences occur. These findings could then be used to refine existing cryopreservation protocols.
Peptide Interactions at Abiotic Surfaces
Another part of Josh’s thesis research focuses on how peptides interact with abiotic, or non-living, surfaces.
Although peptides are often studied in aqueous environments, it is important to develop surfaces that contain active proteins without bulk water. These surfaces may have applications in:
- Textiles
- Medical devices
- Military technologies
Josh currently uses sum frequency generation, or SFG, vibrational spectroscopy to characterize surfaces with chemically immobilized antimicrobial peptides, or AMPs.
SFG is a surface-sensitive technique that can be used to monitor protein secondary structure.
The next goal of this research is to modify the abiotic surface so that the antimicrobial peptides maintain both their proper secondary structure and their antimicrobial activity.
Publications
Khamaladze A, Matz RS, Epstein T, Jasensky J, Banaszak-Holl MM, and Chen Z. Cell volume changes during apoptosis monitored in real time using digital holographic microscopy J. Struct. Biol. 2012, 178(3):270-278.
Ye SJ, Li HC, Wei F, Jasensky J, Boughton AP, Yang P, and Chen Z. Observing a Model Ion Channel Gating Action in Model Cell Membranes in Real Time in Situ: Membrane Potential Change Induced Alamethicin Orientation Change J. Am. Chem. Soc. 2012, 14(134):6237-6243.
Liu Y, Jasensky J, and Chen Z. Molecular interactions of proteins and peptides at interfaces studied by sum frequency generation vibrational spectroscopy Langmuir 2012, 28(4):2113-2121.
Khamaladze A, Jasensky J, Zhang C, Han XF, Ding J, Seeley E, Liu XR, Smith GD, and Chen Z. Hyperspectral microscopic imaging by multiplex coherent anti-Stokes Raman scattering (CARS) Proc. SPIE 2011, 8158, 815805.
Zhang C, Wang J, Khmaladze A, Liu Y, Ding B, Jasensky J, and Chen Z. Examining Surface and Bulk Structures Using Combined Nonlinear Vibrational Spectroscopies Opt. Letts. 2011, 36(12):2272-2274.
Sam Kotler
Research Summary
During my time on the Molecular Biophysics Training Grant, I have studied interactions between beta-amyloid, or A-beta, and lipid bilayers.
Beta-amyloid is an amyloid-forming peptide associated with the pathology of Alzheimer’s disease, or AD. It is believed to play an important role in neuronal toxicity, although the exact mechanism is still not fully understood.
One proposed mechanism is that beta-amyloid binds to and disrupts cell membranes. This disruption may lead to calcium ion uptake, which can trigger an apoptotic, or cell-death, cascade.
Membrane Disruption by Beta-Amyloid
I used fluorescence spectroscopy to show that beta-amyloid disrupts membranes through a two-step mechanism:
- Beta-amyloid oligomers bind to the membrane and form ion-permeable pores.
- Beta-amyloid fibrillization causes membrane fragmentation through a detergent-like mechanism.
This work also showed that gangliosides are key membrane components that mediate the second step of this mechanism. This research was published in Biophysical Journal in June 2012 and forms the basis for my current research.
Current and Future Research
Although this work suggests a mechanism for beta-amyloid toxicity, less is known about specific interactions between beta-amyloid and lipids.
I plan to continue using fluorescence spectroscopy, along with solution-state and solid-state nuclear magnetic resonance, or NMR, spectroscopy. These methods will help characterize the structure and dynamics of beta-amyloid interactions with gangliosides.
My current research focuses on how gangliosides alter the structural morphology of beta-amyloid during early and late stages of amyloid aggregation.
Through this work, I hope to clarify how membrane composition contributes to toxic amyloid formation, with a specific focus on beta-amyloid and ganglioside interactions.
Stephanie Le Clair
Research Summary
During my time on the Molecular Biophysics Training Grant, I used sum frequency generation, or SFG, spectroscopy to monitor how truncated forms of human and rat islet amyloid polypeptide, or IAPP, interact with membranes in real time.
IAPP is the main component of amyloid plaques found on the pancreatic islets of Langerhans in patients with type 2 diabetes.
IAPP Membrane Interaction Studies
My research focused on truncated forms of human and rat IAPP containing residues 1 through 19.
Human IAPP residues 1 through 19 are cytotoxic but do not form fibers. Rat IAPP residues 1 through 19 differ from the human form at only one position, residue 18, but are significantly less cytotoxic.
The goal of this research was to understand why these two peptides differ in cytotoxicity.
Using SFG spectroscopy, I analyzed the amide I band of each peptide to determine:
- Secondary structure
- Membrane orientation
- Differences in membrane interactions
These experiments were performed using membranes with different compositions and several buffer conditions.
I also used Fourier transform infrared-attenuated total reflection, or FTIR-ATR, spectroscopy to validate the peptide orientations measured with SFG.
This work showed differences in the SFG limits of detection when comparing rat IAPP residues 1 through 19 with human IAPP residues 1 through 19.
Current Research
After the training grant ended, I continued my research with support from a National Science Foundation, or NSF, Graduate Research Fellowship and began a new project.
My current research focuses on the structure and dynamics of the full-length cytochrome b5-cytochrome P450, or cyt b5-cyt P450, complex in a membrane environment.
These proteins play important roles in the metabolism of many endogenous and exogenous compounds. However, relatively little is known about how the full-length proteins interact with one another.
Cytochrome b5-Cytochrome P450 Complex
I used solution nuclear magnetic resonance, or NMR, spectroscopy to study complex formation between cyt b5 and cyt P450 in isotropic bicelles. These experiments were performed both with and without a cyt P450 substrate.
This work helped identify the interaction interface on cyt b5 that binds cyt P450.
I used these data, along with double mutant cycle analysis, to model the cyt b5-cyt P450 complex structure using HADDOCK, a computational docking method for biomolecular complexes.
Ongoing Research Questions
Through a series of NMR experiments, we are studying the role of electrostatic interactions in the formation of the encounter complex between cyt b5 and cyt P450.
We have also identified key hydrophobic “hot spot” residues in cyt b5 that are essential for binding to cyt P450.
In addition, we are investigating how cyt P450 ligands affect the interaction between cyt P450 and cyt b5.
Specific regions of cyt b5 and cyt P450 are also being studied to better understand their roles in complex formation, including:
- The linker region of cyt b5
- The transmembrane domain of cyt P450
Publications
Le Clair, S. V.; Zhang, M.; Im, S.-C.; Waskell, L.; Ramamoorthy A. “Anatomy of the cyt b5interface in its stereospecific complex and encounter complexes with cytochrome P450.” In preparation.
Ahuja, S.; Jahr, N.; Im, S.-C., Vivekanandan, S.; Popovych, N.; Xu, J.; Soong, R.; Le Clair, S. V.; Nanga, R. P.; Yamamoto, K.; Bridges, A.; Waskell, L.; and Ramamoorthy, A. “A dynamic structure of the membrane-bound cytochrome b5-cytochrome P450 complex from NMR and mutagenesis data.” Submitted.
Nguyen, K.; Le Clair, S. V.; Ye, S.; and Chen, Z. “Orientation Determination of Protein Helical Secondary Structure Using Linear and Nonlinear Vibrational Spectroscopy.” J. Phys. Chem. B.2009, 113, 12169-12180.
Nguyen, K.; Le Clair, S. V.; Ye, S.; and Chen, Z. “Molecular Interaction between Magainin 2 and Model Membranes in Situ.” J. Phys. Chem. B. 2009, 113, 12358-12363.
Ye, S.; Nguyen, K. T.; Le Clair, S. V.; and Chen, Z. “In Situ Molecular Level Studies on Membrane Related Peptides and Proteins in Real Time Using Sum Frequency Generation Vibrational Spectroscopy.” J. Struct. Biol. 2009, 168, 61-77.
Le Clair, S. V.; Nguyen, K.; and Chen, Z. "Sum Frequency Generation Studies on Bioadhesion: Elucidating the Molecular Structure of Proteins at Interfaces.” J. Adhesion. 2009, 85, 484-511.
Esmonde-White, K. A.; Le Clair, S. V.; Roessler, B. J.; Morris, M. D. “Effect of Conformation and Drop Properties on Surface-Enhanced Raman Spectroscopy of Dried Biopolymer Drops.” Appl. Spectrosc. 2008, 62, 503-511.
Anton Loukianov
Research Summary
While on the Molecular Biophysics Training Grant, I have had the opportunity to explore two distinct areas of research: computational neuroscience and experimental ultrafast spectroscopy.
Computational Modeling of Neuronal Networks
My first project took place during a rotation in Dr. Zochowski’s lab. I used computational models to study networks of neurons.
The goal of this research was to better understand how neurons form connections during brain development.
To make the problem more manageable, we used a Kuramoto harmonic oscillator as a simplified model of a neuron. We then studied the behavior of a neuronal network in which each neuron had distance-dependent positive and negative coupling functions.
Using this model, we explored which combinations of parameters produced different network behaviors, including:
- Oscillating modes
- Traveling waves
- Random oscillations
Through this project, I studied:
- Dynamical systems theory
- Modeling in the C plus plus programming language
- Large-scale computations using a computing cluster
Ultrafast Two-Dimensional Spectroscopy
After that rotation, I realized that I preferred experimental work. I then joined Dr. Ogilvie’s ultrafast two-dimensional, or 2D, spectroscopy group.
My current project focuses on developing an experimental setup to study the Stark shift in two-photon absorption, or 2PA, spectra of green fluorescent protein, or GFP, derivatives.
Recent research has reported that 2PA spectra are strongly dependent on local electric fields and should exhibit large Stark shifts. I aim to use this dependence to develop optical probes that can measure electric fields in biological samples.
Potential Applications
An optical electric-field probe could make it possible to:
- Observe electric fields in firing neurons at biologically relevant timescales
- Measure pH using a membrane-embedded fluorophore
- Perform high-resolution imaging using field enhancement, potentially in combination with two-photon fluorescence
Research Training
To support this work, I have:
- Built electronics to control a high-voltage power supply
- Designed and built sample holders
- Performed preliminary measurements on samples
I have also received training in:
- Ultrafast optical setups
- Machine shop techniques
- Cryogenics
- Basic wet lab techniques
My coursework in nonlinear optics, quantum mechanics, statistical mechanics, and biophysics supports my ability to analyze and interpret my experimental data.
Seth McCubbin
Research Summary
Seth McCubbin’s research focuses on the dynamic behavior of microtubules. Microtubules are intracellular polymers that play important roles in:
- Cell division
- Cell motility
- Intracellular transport
Microtubules are assembled from alpha-beta tubulin subunits. Their assembly is governed by a process called dynamic instability, in which microtubules abruptly switch between phases of slow growth and relatively rapid shortening.
Proper microtubule function depends on these unique dynamics. These dynamics can be affected by cellular proteins, such as tau, and by drugs, such as taxol.
Tau is a protein associated with Alzheimer’s disease and is thought to stabilize microtubules. Taxol is a chemotherapy drug that affects the ability of microtubules to coordinate chromosome segregation. However, the effects of these molecules at therapeutic concentrations are not fully understood.
Seth’s research aims to study how substoichiometric binding of taxol and tau affects microtubule kinetics.
Taxol and Microtubule Dynamics
Early taxol experiments used differential interference contrast, or DIC, microscopy to collect polymerization data at therapeutic taxol concentrations ranging from zero to 100 nanomolar.
More recent data have been collected using total internal reflection fluorescence microscopy, or TIRFM. This method improves both spatial and temporal resolution.
Using TIRFM, Seth can measure microtubule length changes within 25 nanometers at 8 hertz. This technique also makes it possible to detect changes in microtubule tip structure.
Research Findings
Results show that taxol strongly affects microtubule growth at concentrations as low as 10 nanomolar.
Compared with control conditions, taxol-treated microtubules show greater variability in growth rate, especially over longer timescales of about 100 seconds.
In some cases, microtubules switch between normal growth and periods of very slow growth.
Additional findings include:
- Taxol at 10 nanomolar almost completely suppresses rapid shortening.
- Beginning at 100 nanomolar taxol, tubulin on-rates and off-rates gradually decrease.
- The net growth rate remains constant when periods of very slow growth are excluded.
Future Research
Future work will investigate how tau affects microtubule dynamics.
Seth also plans to confirm these results using optical tweezers. This approach will allow microtubule polymerization to be studied at nanometer and millisecond resolution.
Stephen Norris
Research Summary
Stephen Norris studies how materials are transported inside cells.
Cells are highly complex systems that are regulated across both space and time. Subcellular cargoes, including organelles, endosomes, lysosomes, and messenger RNA, or mRNA, granules, are often directed to specific locations within the cell at specific times.
This targeted intracellular trafficking is usually carried out by molecular motor proteins, such as kinesin and dynein. These proteins move cargo along cytoskeletal tracks.
Groups of multiple motors working together are thought to be necessary to generate enough force and travel distance to move large cargoes through the dense environment of the cytoplasm.
Background
Over the past two decades, highly precise single-molecule experiments have shown how individual kinesin or dynein motors generate force in vitro, or outside of a living cell.
However, studying multiple-motor cargo transport in live cells is difficult because of the complexity of the cellular environment.
Multiple-motor experiments performed in vitro can also be highly artificial and may not provide ideal experimental precision.
Research Approach
Stephen’s research aims to connect these two types of experiments by developing a synthetic biology method to create well-characterized multiple-motor complexes.
This approach uses a protein-based method to attach known numbers of motor proteins to scaffolds with known length and flexibility.
These custom-designed constructs are expressed in mammalian COS-7 cells, where they assemble into multiple-motor complexes inside living cells.
The movement of these complexes can then be studied:
- In live cells
- In vitro
- Using total internal reflection fluorescence, or TIRF, microscopy
Research Impact
This method allows researchers to investigate important questions in the molecular motor field with high accuracy and precision.
Although Stephen is applying these synthetic biology methods to molecular motors, the same tools could also be used more broadly. For example, they could help create multi-protein complexes in live cells when genetic fusion methods are not sufficient.
Publications
(In preparation – Biophysical Journal): J.A. Cribb, P.A. Vasquez, P. Moore, S. Norris, S. Shah, M.G. Forest, and R. Superfine. Nonlinear signatures of entangled polymer solutions in active microbead rheology.
Andrew Sikkema
Research Summary
Andrew Sikkema studies how natural products are made and how this knowledge could support the development of new or improved medicines.
Natural products make up a large portion of medicinal compounds used today. Understanding how these compounds are produced could improve researchers’ ability to create new drugs or modify existing ones.
Many natural products are made through polyketide synthase, or PKS, pathways, or through mixed PKS-nonribosomal peptide synthetase, or PKS-NRPS, pathways. These biosynthetic pathways are found in organisms ranging from archaea to animals.
These pathways are organized into modules. Each module contains multiple domains that add or modify a building block on a growing molecule. This process is similar to an assembly line, where parts are added step by step.
By rearranging or modifying these modules, organisms have evolved pathways that produce many different compounds. Understanding how these modules function and interact with one another is an important step toward engineering pathways that can modify natural products or create new compounds.
Apratoxin Pathway Research
During his time on the Molecular Biophysics Training Grant, Andrew’s research has focused on the apratoxin pathway, which produces the macrolactone compound apratoxin.
Apratoxin is found in a marine cyanobacterium. The apratoxin pathway is a useful system for studying how natural product synthesis begins.
Apratoxin has also been shown to selectively target cancerous cells, making this research potentially valuable for medical applications.
Biosynthesis Initiation
Synthesis in the apratoxin pathway begins with the first module, called the loading module.
In this pathway, the loading module does two important things:
- It initiates synthesis.
- It produces a unique tert-butyl group.
To understand this process, Andrew uses structural biology methods, including:
- X-ray crystallography
- Electron microscopy
This structural work is supported by biochemical assays that track:
- The initiation of biosynthesis
- Modification of the substrate
High-Throughput Screening
As a complementary part of this work, Andrew has collaborated on the development of a high-throughput screening method using biolayer interferometry.
This technique is used to test multiple protein constructs under different lysis conditions. The goal is to identify conditions that maximize protein yields.
Zachary Stein
Research Summary
My research uses nuclear magnetic resonance, or NMR, spectroscopy to better understand the dynamics of ribonucleic acid, or RNA, and deoxyribonucleic acid, or DNA.
Specifically, I use an NMR method called relaxation dispersion. This technique can help detect and characterize different structural states that RNA and DNA molecules adopt over time.
RNA and DNA Dynamics
Recent evidence suggests that RNA and DNA molecules often sample two or more structural states inside the cell.
Studying these states can help researchers better understand:
- How DNA is regulated
- How RNA carries out cellular processes
- How biomolecular structure changes over time
Biomolecules usually spend most of their time in a ground state. In this state, different biomolecules may appear similar to one another.
However, biomolecules can also spend a smaller amount of time in excited states. These excited states may have more distinct shapes. Understanding these unique shapes could help researchers design drugs that bind more specifically to their intended biomolecular targets.
This approach may support the development of safer and more effective drugs. Many drug side effects occur because drugs affect multiple cellular processes, rather than one specific target.
Multi-State NMR Analysis
Current methods are effective for modeling and detecting RNA or DNA molecules that exchange between two physical states.
However, systems with three or more states are more difficult to detect. These multi-state systems can be mistaken for two-state systems, especially when data quality is limited.
My research focuses on developing alternative data analysis methods to better distinguish among the factors that influence molecular exchange. These methods may improve our understanding of RNA and DNA dynamics.
Research Impact
There is limited literature on the theory of NMR for multi-site exchange. For this reason, success in this work could support both my own research and the broader research community.
The goal is to provide a useful technique for other researchers who study biomolecular dynamics using NMR.
Matt Stone
Research Summary
My research explores how B lymphocyte antigen receptor, or BCR, signaling uses lipid organization to regulate cellular signaling.
BCR signaling is a key process in B cells, which are immune cells involved in recognizing antigens and coordinating immune responses.
Lipid Organization and BCR Signaling
Lipid phase separation has been studied extensively in model membranes. However, there is limited direct evidence showing that lipid organization exists and has a functional role in living cells.
Many models of BCR signaling propose that lipid organization helps regulate how antigen binding is communicated across the plasma membrane. These models suggest that lipids may control whether specific membrane proteins move into regions near clustered BCRs.
My goal is to clarify how lipid-mediated heterogeneity contributes to BCR signal regulation.
Super-Resolution Imaging Approach
To study this process, I use two-color super-resolution microscopy.
I have applied highly accurate image registration methods to our lab’s existing super-resolution imaging techniques. This allows for accurate two-color, single-molecule microscopy.
Using this approach, I have observed lipid reorganization that depends on BCR antigen binding.
These observations show that branched and saturated lipids respond differently to BCR clustering. This behavior is consistent with lipid phase behavior observed in model membrane systems.
Research Impact
Understanding how lipid organization affects BCR signaling may support the development of new treatments for B cell-based autoimmune diseases and cancers.
In particular, this work may help identify new drug strategies that target lipids involved in immune cell signaling.
Method Development
In addition to studying BCR signaling, I am characterizing the fluorescence properties of indocarbocyanine dyes for use in quantitative two-color super-resolution microscopy.
I am also developing new analysis methods based on correlation functions. These methods will support accurate measurements of spatial and temporal correlations between separately labeled biomolecules.
I plan to share these methods through publications and conferences so that other research groups can use them in related studies.
Publications
Chen, Ran, Tatsiana A Ratnikova, Matthew B Stone, Sijie Lin, Mercy Lard, George Huang, JoAn S Hudson, and Pu Chun Ke. 2010. “Differential uptake of carbon nanoparticles by plant and Mammalian cells.” Small (Weinheim an Der Bergstrasse, Germany) 6 (5) (March 8): 612-617.
Veronica Taylor
Research Summary
This research focuses on how RhoA, a member of the Ras family of guanosine triphosphatases, or GTPases, is activated through cell signaling pathways.
RhoA is an important regulator of the actin cytoskeleton, which helps control cell shape, movement, and organization.
RhoA Activation Pathway
RhoA can be activated in response to G protein-coupled receptor, or GPCR, signaling through G alpha q, also written as Gαq.
When activated G alpha q binds to the effector protein p63RhoGEF, p63RhoGEF changes into an active conformation. In this active state, p63RhoGEF shows RhoA-specific guanine nucleotide exchange factor, or GEF, activity.
GEF activity helps activate RhoA by promoting nucleotide exchange.
Research Question
The mechanism by which G alpha q activates p63RhoGEF is still unknown.
The crystal structure of p63RhoGEF in its activated conformation has been determined. However, attempts to crystallize the basal, inactive conformation have not been successful.
Additional biochemical studies have also not fully explained how p63RhoGEF autoinhibition is relieved when it binds G alpha q.
Research Approach
We are using nuclear magnetic resonance, or NMR, spectroscopy to study the p63RhoGEF catalytic core in solution.
This approach will help us:
- Determine the solution structure of p63RhoGEF in its basal conformation
- Identify residues that undergo large conformational changes after activation by G alpha q
- Better understand how p63RhoGEF is regulated
Research Impact
Successful completion of this work would provide a molecular explanation for how p63RhoGEF is regulated by G alpha q.
This research would also clarify an important connection between heterotrimeric G proteins and small molecular weight G proteins.
