2015-16 GAANN and COS Molecular Biophysics Students
2014-15 GAANN and COS Molecular Biophysics Students
Molecular Biophysics Trainees are drawn from the Schools of Applied Physiology, Biology, Chemistry & Biochemistry, Physics, and the College of Engineering. Trainees participate in the weekly Research Review and a laboratory rotation program. Interdisciplinary training in biophysics prepares students for cutting-edge research at the interface of the physical and biological sciences.
Gold nanoparticles have been increasingly used for biomedical applications, including imaging, drug delivery, and photothermal cancer therapy. The high surface to volume ratio of nanoscale materials allows for the loading of many drug molecules or targeting ligands onto each nanoparticle while using minimal gold concentrations, which helps to reduce deleterious side effects. The plasmonic properties of the gold nanoparticles, namely their strong light scattering and absorption properties, also make them highly suitable as imaging probes. To improve the design of gold nanoparticles for these applications, significant work has been done to better understand how varying their physical and surface chemical properties affect their interactions in biological environments. For example, the size of nanoparticles has been shown to influence their cellular uptake, with ~30 – 40 nm gold nanoparticles showing the highest rates of endocytosis. The surface chemistry (e.g. targeting peptides, proteins, drug molecules, etc.) of nanoparticles has also been shown to affect their rate of localization within cells and their toxicity. However, little is known about the interaction of nanoparticles with the cell nucleus, which stores genetic information and controls the life and death of the cell. This work will study how the size and shape of nuclear-targeted gold nanoparticles affects their uptake into cell nuclei, their interactions with DNA, and their cytotoxicity. Ultimately, the level of nuclear uptake will be correlated to the effectiveness of treatments such as drug delivery or photothermal cancer therapy, which relies on rapid heat production from localized gold nanoparticles to induce apoptotic cell death.
Biomaterials scientists have explored controlling stem cell fate through mimicking the cellular microenvironment using synthetic materials in combination with naturally derived cell adhesive peptides. Understanding the extracellular environment is key to providing insight into stem cell adhesion, spreading, differentiation, and proliferation. The common extracellular matrix (ECM) proteins such as fibronectin (Fn), fibrin, collagen, and laminins are known to bind to cells and direct cell fate via interaction with cell surface receptors known as integrins. These cell-ECM interactions are complex and influence factors including surface activation and/or protein conformation. Many groups, including our own, have attempted to direct integrin-binding and cellular responses through rational design of engineered protein fragments. We have utilized Fn fragments that display stabilized conformations in order to promote binding of certain integrins over others. Though useful, this approach is limited by current knowledge of structure/function relationships of ECM proteins and integrins. I'll seek to generate novel peptides that may be able to better mimic the cues a cell encounters in a specific environment through creation of a library of evolutionary variants from a scFV yeast display library. The idea involves combining the integrin binding domain of Fn with those antibody fragments which should facilitate the discovery of new ECM-inspired protein domains that trigger specific cellular responses. This library will be designed based on the scFv library and the integrin binding domain. Subsequently, presentation to desired mammalian cells will occur in order to screen a high-throughput system that enables the exploration of cell differentiation, spreading, adhesion, and proliferation.
Many Gram-negative bacteria are highly virulent and are becoming increasingly resistant to modern antibiotics due to improper use. Drug uptake in Gram-negative bacteria is challenging due to the presence of an asymmetric outer membrane (OM) that hinders uptake and numerous efflux pumps in the cytoplasmic membrane, which expel antibiotics that do enter the cell. While small molecules (< ~600 Da) can diffuse through OM porins, large molecules, nutrients and antibiotics require the inner membrane’s proton motive force and integral membrane proteins to facilitate their active transport. Potential antibiotic targets on the OM include the TonB-dependent transporters (TBDTs) that, true to their name, employ the periplasm-spanning TonB protein to import large, rare nutrients such as ferric iron siderophores, nickel chelates, and cobalamins into the periplasm. Characteristic of all TBDTs is a luminal domain, which prevents the passive diffusion of molecules into the periplasmic space, but needs to be unfolded to permit nutrient import. Through the use of molecular dynamics simulations, we seek to understand the OM import pathway of TBDTs from nutrient binding to luminal domain opening and eventually nutrient import and luminal domain refolding.
Cu/Zn Superoxide Dismutase (SOD1) is an abundant protein and highly conserved across the eukaryotic domain. Surprisingly, only a minimal amount of SOD1 is required to prevent intracellular oxidative stress, which begs the question: What is the purpose for its large excess? We propose that a major role of SOD1 in cell biology is to mediate redox-signaling cascades through its ability to disproportionate superoxide into oxygen and hydrogen peroxide, the latter of which is a well-known redox signal. Recent studies show that the peroxide generated by SOD1 is able to stabilize a membrane-tethered yeast casein kinase (YCK1). This stabilization leads to a metabolic switch from respiration to fermentation, which is the favored mode of metabolism for cancerous cells. Our results between SOD1 and casein kinase were observed in mammalian cell lines as well, but the mechanistic details are unknown for both cells types. Therefore, this research has the potential to further our current knowledge of cancer cell metabolism. Through a proteomics approach and a genetic screen, we will elucidate the mechanism of YCK1-SOD1 stabilization in yeast, while studying the homologous, mammalian Wnt signaling pathway in parallel. We anticipate our studies will uncover new and exciting aspects of the redox biology of SOD1 that go well beyond its role in oxidative stress protection.
Phenylketonuria (PKU) is a genetic disease that leads to an accumulation of the amino acid phenylalanine in the blood, resulting in stunted growth, seizures, and cognitive impairment. Some moderate forms of PKU respond to tetrahydrobiopterin (BH4) supplementation, which lowers the levels of phenylalanine in the blood, and are known as BH4-responsive PKU. Today, chemically synthesized BH4 is used to treat BH4-responsive PKU and is the only source of therapeutic BH4 in the US. Our goal is to engineer the biological manufacturing of BH4from glucose using Saccharomyces cerevisiae as the microbial host, resulting in the isolation of sufficient microbially produced BH4 for complete chemical characterization and future in vivo testing.
The copper (I) catalyzed 3+2 cycloaddition, or CuAAC, reaction between an azide and a terminal alkyne is stereospecific, produces no byproducts, and is tolerant of a wide variety of functional groups and solvent systems. Additionally, the reaction does not proceed in the absence of the copper catalyst. The CuAAC reaction is therefore an ideal tool for gathering information about biological systems by enabling the specific connection of biomolecules when and where desired. The reaction components do not engage in side reactions with other chemicals in biological environments and can be easily inserted into cellular systems to serve as ‘handles’ to label proteins in real time. However, the full potential of the reaction cannot be realized in living systems because free copper ions are toxic in cells. We therefore seek to discover biocompatible peptides that can bind copper ions and catalyze the CuAAC reaction using native copper already present in cells. To identify such ligands, one-bead one-compound libraries of oligopeptides will be constructed to provide a high-throughput synthesis and testing of candidate catalyst sequences. After identification of the most active sequences, follow-up study and optimization will focus on copper binding ability, performance in complex media and cellular expression of the peptide. Ultimately, biocompatible CuAAC reactions will allow scientists to explore a wide variety of molecular processes in living cells in real time.
Life is a master stenographer—writing, rewriting and recording its history in elaborate biological structures. The keys to unlock the origin of life lie encrypted in the ribosome, the oldest and most universal assembly of life. The ribosome represents the pivotal point at which chemical systems began biological evolution by converting information in mRNA into functional proteins. We have previously described an atomic level evolutionary model of the ribosome, grounded in patterns of ribosomal RNA growth in relatively recent ribosomal expansions, for which there is an extensive, atomic-resolution record. Using this model, we have retraced the earliest evolution of the ribosome on an early Earth. By an iterative hypothesis-testing process, we computationally and experimentally recapitulate a feasible early evolution of the ribosome, creating testable molecular models of ancestral ribosomal components. These model ancestral molecules are evaluated by iterative computation and experiment, including folding, assembly and catalysis assays, and association with ancient peptides and proteins. Folding and assembly experiments include chemical footprinting, spectrophotometry and gel-shift assays. Catalytic competence is quantified by the peroxidase assay, and thermodynamic minima and effects of co-assembly on thermal stability are determined by spectrometric melting. Through this series of experiments we further characterize the oldest traceable macromolecules and uncover clues and key steps that connect the putative RNA world to the RNA-DNA-protein world of extant life.
Gram-negative bacteria possess two membranes, the inner and outer of which contain primarily α-helix and β-barrel proteins respectively. In recent years, significant progress has been made in understanding insertion and assembly of proteins into the inner membrane, while the same process in the outer membrane has remained elusive. In 2013, the crystal structure of BamA, the central and essential component of the β-barrel assembly machinery (BAM), was released, paving the way for rapid progress in understanding the insertion and assembly process. All-atom molecular dynamics simulations have been performed, revealing many novel features including lateral gate opening between the first and last barrel strands, and a significantly thinner, destabilized membrane region near the putative insertion site. However, many questions remain, including the role of the periplasmic domains, the mode of substrate recognition, and the energetic factors driving function in the absence of both ATP and an electrochemical gradient. Currently, we are performing novel equilibrium simulations of the protein in its native membrane environment. In addition, we are calculating the free energy of lateral-gate opening for native systems, as well as systems with strand modifications and augmentations designed to yield insight into driving energetics and substrate recognition
Long distance electron transfer (ET) plays a crucial role in many important biological processes. Rapid electron transport in proteins is facilitated by step-wise hopping between aromatic amino acid side chains. In some cases, this phenomenon is photoinitiated. For example, in DNA photolyase and cryptochrome, electron transfer is mediated via a triad of tryptophan residues. In photosystem II, a photosynthetic reaction center, a tyrosine acts as an essential cofactor in the light-driven oxidation of water via a manganese-calcium cluster. To model the effects of aromatic interactions on electron transfer, we have constructed beta-hairpin maquettes containing tyrosine and tryptophan. The physiochemical properties of these maquettes are investigated using a combination of techniques, including differential pulse voltammetry (DPV), circular dichroism (CD), electron paramagnetic resonance (EPR), UV resonance Raman (UVRR), and transient absorption (TA) spectroscopy. This work will elucidate the mechanism by which electron transfer is thermodynamically and kinetically controlled in biologically relevant models. Understanding these parameters will inform biomimetic strategies in artificial solar energy conversion.
Membrane proteins occupy a variety of functional niches in the cell including structural support, regulation, and communication; understanding how these proteins accomplish their role in the cell can be enhanced with the knowledge of the membrane protein structure. One method of membrane protein structure determination is via 2D crystallography. In this method, solubilized membrane proteins are crystallized in lipid membranes via manipulation of the dialysis conditions, like lipid to protein ratio (LPR), salt type and concentration, dialysis length, lipid type and concentration, etc. As these conditions are changed, 2D crystals are screened for via uranyl acetate negative staining and imaging with a transmission electron microscope. Once crystallization conditions are optimized for membrane formation, high resolution images are taken via electron cryo-microscopy (cryo-EM). A crystal structure is determined with the help of image processing. Both membrane bound and membrane associated membranes can be studied via 2D crystallography. Understanding the structure of membrane proteins can lead to insight on how proteins function within the cell.
Large data sets can be analyzed and inherent patterns extracted using the discriminatory power of robust statistical algorithms developed in our lab. These methods deconstruct complex, multidimensional data into true linear distances among samples. Applied to rapidly characterizing antibiotic resistant bacteria, various bacterial samples are interrogated for optical changes via flow cytometry. Both scatter signatures, and fluorescence are measured and changes relative to paired controls directly and rapidly report on antibiotic susceptibility in complex media. For instance, classifying bacteria in the blood stream is an area of clinical importance for this technology. Current clinical standards take upwards of 48 hours to classify bacteria and determine susceptibilities. Our goal is to cut this time to obtain actionable treatment information by half, to improve patient outcomes.
Asthma is the most common chronic illness of childhood, and the prevalence of this airway disease has been increasing since the early 1980s. Excessive levels of reactive oxygen species (ROS), termed oxidative stress, in airways of asthmatic patients positively correlate with disease severity. However, the cellular and molecular mechanisms by which oxidative stress leads to these symptoms of asthma remain poorly understood. T cells are known to play a central role in the development and maintenance of asthma. It is established that various signaling molecules within T cells, that are important for activation and function, are ROS-sensitive. In asthmatic patients, there is an increased number of effector T cells and these T cells are more functionally active than those in healthy patients. While severe oxidative stress is known to lead to cell growth arrest, apoptosis, or even necrosis, it has been suggested that low levels of cytoplasmic ROS are necessary for T cell activation. We are focusing on the T cell receptor machinery, which is involved in early T cell signaling, since the effects of oxidative stress can occur at small time scales. We are measuring the dynamic kinetics of interactions between molecules necessary for T cell activation in response to ROS using our micropipette-based techniques. Additionally, we are quantifying the spatio-temporal organization of these molecules in response to ROS using our various microscopy techniques. Ultimately, we aim to fundamentally understand how oxidative stress modulates T cell behavior at early time scales.
The volumetric heating values of today’s biofuels are too low to power energy-intensive aircraft, rockets, and missiles. Recently, pinene dimers were shown to have a volumetric heating value similar to that of the tactical fuel JP-10. To provide a sustainable source of pinene, we engineered Escherichia coli for pinene production. We combinatorially expressed three pinene synthases (PS) and three geranyl diphosphate synthases (GPPS) with the best combination achieving ~28 mg/L of pinene. We speculated that pinene toxicity was limiting production; however, toxicity should not be limiting at current production levels. Because GPPS is inhibited by geranyl diphosphate (GPP), and to increase flux through the pathway, we combinatorially constructed GPPS-PS protein fusions. The Abies grandis GPPS-PS fusion produced 32 mg/L of pinene, a 6-fold improvement over the highest titer previously reported to be produced by engineered E. coli. Finally, we investigated the pinene isomer ratio of our pinene-producing microbe and discovered that the isomer profile is determined not only by the identity of the PS used, but also by the identity of the GPPS with which the PS is paired. We demonstrated that the GPP concentration available to PS for cyclization alters the pinene isomer ratio.
The interface between electrical and biological systems mediates the interaction of the human body with modern technology. Current applications include neural probes, bionic implants, and regenerative medicine. However, external probes and bionic devices are often rejected by the body and lack long-term electrical stability. My research, in the Payne Lab in the School of Chemistry and Biochemistry, investigates the use of electrically conductive polymers as stable bioelectrical interfaces to interact with single cells. Conductive polymers have excellent potential to mediate bioelectrical communication due to their high conductivity, mechanical flexibility, and biocompatibility. Additionally, electrically conductive polymer nanowires can be grown between two electrodes by applying an alternating voltage. It is hypothesized that this method can be extended to grow electrically conductive polymer nanowires inside individual mammalian cells between two membrane-inserted electrodes. Further work will aim to extend nanowires across the cell membrane to be accessible to the extracellular environment. These nanowires can then be used as stable and biocompatible connections to electrically interact with single cells. Conductive nanowire connections to individual cells would create a seamless interface for bionic implants and provide a less invasive approach for single neuron voltage recordings in the brain.
Protein crystallography involves packing identical macromolecules in a highly ordered lattice to obtain high-resolution information on the overall protein structure. Membrane proteins can be difficult for crystallographers because they consist of hydrophobic segments that are insoluble in water. Removing them from their native membrane can lead to instability. However, by using detergents to solubilize these hydrophobic segments, one can isolate and purify membrane proteins. Dialysis is then performed to remove the detergent and allow the protein to reconstitute back into a lipid bilayer. If this protein reconstitutes in ordered arrays, it creates a two-dimensional crystal, which can be further processed to determine the structure. I am studying the structure and function of a macromolecular complex, which has implications in alternative energy. By following these steps towards crystallization and structure determination, I can answer many questions about the system and inspire new strategies towards energy development.
Phenotypic diversification of genetically identical cells arises from variable mRNA level. Hence, quantification of mRNA transcript number at the single cell level is important for understanding the origin of stochastic gene expression. The mRNA level of individual cells can be measured using Fluorescence In Situ Hybridization (FISH), which has been widely adopted for a variety of model organisms. This method depends on covering a long region of the target mRNA with multiple fluorescently labeled DNA probes with tens of fluorescent dyes. Here, we introduce a FISH protocol for detection of mRNA with a short singly labeled DNA probe in budding yeast. Using highly inclined laser illumination, we achieve single fluorophore sensitivity across a wide range of transcript levels. We also achieve excellent hybridization specificity between probe and target mRNA sequence and improved signal to noise ratio using methanol fixation followed by zymolyase treatment. Our time and cost efficient method has potential applications for detection of short mRNA in higher organisms.
Ribonucleotide reductase (RNR) produces deoxynucleotides from nucleotides via a conserved radical transfer pathway in the α2β2 quaternary complex. The reaction is initiated using a stable tyrosyl radical in the β2 subunit. Nucleotide reduction takes place 35Å away in the active site of the larger α2 subunit. Radical transfer occurs through a hopping mechanism along a pathway of conserved aromatic amino acid residues into the RNR active site. RNR inhibition has been a major focus in cancer treatment. Hydroxyurea (HU) inhibits RNR, in what has been proposed to be a radical quenching mechanism. The mode of HU inhibition is of great import in the design and application of new anti-cancer therapeutics. We are using Fourier transform infrared (FTIR) spectroscopy to detect single amino acid changes that take place as a result of RNR inhibition with HU and other compounds.
