Biochemical and Biophysical Systems Group
The Biochemical and Biophysical Systems Group is formed by experimental and computational biologists who use a wide range of expertise to approach cutting-edge problems in systems biology. We use multidisciplinary approaches – ranging from molecular biology through proteomics to modeling – to investigate microbes and microbial communities as they respond to different perturbations, including those relevant to emerging issues in bioenergy and pathogenesis. In addition to developing computational tools to describe and predict biological systems, we are combining experimental efforts with modeling and simulation methods to design and develop safe and effective therapeutics. Our principal unifying objective is to gain a predictive understanding of protein-mediated activities that are critical to cells and their interactions in living systems.
Contact: Fang Qian
Nanowires may be extremely useful for commercial applications such as flexible displays, solar cells, catalysts, and heat dissipators. Long nanowires can be efficiently "grown" in solution, but the process also yields undesirable byproducts: nanoparticles and short rods.
A new method uses hydrophobic interactions to allow the desired nanowires to self-separate from the solution to an immiscible organic solvent. Like oil in water, the organic solvent forms droplets -- the nanowires cross the solvent-water interfaces.
The key is the "capping agent." This molecule binds preferentially to the side crystal facets of the growing nanowire, blocking growth there while allowing crystal growth at the nanowire ends. The use of a hydrophobic capping agent makes the self-separation of the nanowires into the organic solvent energetically favorable compared to those hydrophobic surfaces remaining in the aqueous solution. The nanoparticles and short rods are less likely to come in contact with the organic-solvent droplets, and surface tension also hinders the nanowires' crossing into the solvent.
The organic-solvent/purified nanowire phase settles out of the solution (or can be centrifuged out), completing the purification process.
See Multiphase Separation of Copper Nanowires, Fang Qian, Pui Ching Lan, Tammy Olson, Cheng Zhu, Eric B. Duoss, Christopher M. Spadaccini, and T. Yong-Jin Han, Chem. Commun., 2016, 52, 11627.
Contact: Fang Qian
Supercapacitors with energy storage far beyond that of advanced batteries would be possible with increases in energy density. This new manufacturing technique provides for such advances.
Supercapacitors depend on an electrolytic ion double-layer and/or ion-electrode charge-transfer ("pseudocapacitive") reactions on the surface of an electrode. The more surface area the better.
Highly porous and conductive graphene aerogels, which have very high surface-area-to-bulk ratios, would seem a good choice for a supercapacitor electrode. The pores, however, are too tortuous and poorly connected for electrolytic ions to function effectively. Thin electrodes are good for reducing the overall mass of the device, but are limited in their "active mass" -- their accessible surface. In addition, thin electrodes are fragile and thus difficult to make and assemble into working devices.
The 3D-printed micro-structured graphene aerogel provides significantly enhanced access to the electrode for the electrolyte, even with a thicker, easier-to-assemble electrode. A new graphene oxide-based ink compatible with a modified "direct-ink" 3D printer enables this innovation.
Graphene-based electrodes have the advantage that graphene is highly conductive; this saves the weight and volume of the separate conductive layer in conventional supercapacitor electrodes. The electrode created in this work also has enhanced conductivity due to the inclusion in the aerogel source material of fumed silica particles as sacrificial templates to produce pores and graphene nanoplatelets.
The resulting supercapacitor is extremely stable (over more than 10,000 charge/discharge cycles) and has exceptional capacitive retention with power densities that equal or exceed those of devices made with electrodes that are 10 to 100 times thinner. The thicker, electrolyte-accessible electrodes of the new device allow these power densities to be achieved in much larger-scale devices, offering a route to practical alternatives to conventional batteries in mobile phones, for example.
See Supercapacitors Based on Three-Dimensional Hierarchical Graphene Aerogels with Periodic Macropores, Cheng Zhu, Tianyu Liu, Fang Qian, T. Yong-Jin Han, Eric B. Duoss, Joshua D. Kuntz, Christopher M. Spadaccini, Marcus A. Worsley, and Yat Li. Nano Lett. (2016).
Contact: Ed Lau
Blast-induced traumatic brain injury (TBI) from improvised explosive devices is the most frequent wound occurring from the conflicts in Afghanistan and Iraq. Estimates suggest more than 200,000 veterans have had at least one traumatic brain injury.
Clinical reports and in vivo studies show exposure to a blast can cause TBI, although how the energy is transmitted to the brain is not well understood.
Molecular dynamics (MD) simulations show that ion channels in cell membranes are resistant to damage by shock waves. But when voids ("bubbles") are present, for example, in the extra-cellular fluid, shock-wave-induced void collapse creates nanojets that could cause significant damage to the ion channel. Such damage can contribute to an electrolyte imbalance within nerve cells that can lead to the initial symptoms of TBI, such as headaches and seizures.
Previous experiments and molecular simulations of void-shock interactions have shown that the force generated by void collapse is great enough to cause pores to form in membranes, known as poration. Such pores, however, quickly self-heal, likely avoiding unregulated ion exchange through the membrane that can lead to a cascade of events that cause injury and ultimately neuronal death.
But shock-wave-induced void collapse can have adverse effects on membrane-bound ion channels even when poration does not occur. In contrast to the effects of poration on a simple (lipid-only) membrane, the damaged ion channels may not readily self-heal.
See Shock Wave-Induced Damage of a Protein by Void Collapse, E. Y. Lau, M. L. Berkowitz, E. Schwegler (2016) . Biophysical Journal, 110, 1, 147–156.
Contact: Fang Qian
Programmable positioning of 2 µm polystyrene (PS) beads with single particle precision and location selective, “on-demand”, particle deposition is demonstrated by utilizing patterned electrodes and electrophoretic deposition (EPD). An electrode with differently sized hole patterns, from 0.5 to 5 µm, is used to illustrate discriminatory particle deposition events based on voltage and particle-to-hole size ratio. With decreasing patterned hole size, a larger electric field is required for a particle deposition event to occur in that hole. For a 5 µm hole, particle deposition begins to occur at 10 V/cm whereas an electric field of 15 V/cm is required for particles to begin depositing in 2 µm holes. The likelihood of particle depositions continues to increase for smaller-sized holes as the electric field increases. Eventually, a monolayer of particles begins to form at approximately 20 V/cm. In essence, there is a voltage threshold for each hole pattern of different sizes, allowing fine adjustments in pattern hole size and voltage to control when a particle deposition event takes place, even with the patterns on the same electrode. This phenomenon opens a route toward controlled, multi-material deposition and assembly onto substrates without repatterning of the electrode or complicated surface modification of the particles. An analytical approach using the theories for electrophoresis and dielectrophoresis finds the former to be the dominating force for depositing a particle into a patterned hole. E-beam lithography is used to pattern spherical holes in precise configurations onto electrode surfaces, where each hole accompanies a polystyrene (PS) particle placement and attachment during EPD. The versatility of e-beam lithography is used to create arbitrary pattern configurations to fabricate particle assemblies of limitless configurations, enabling fabrication of unique materials assemblies and interfaces.
SEM images showing the effect of pattern hole size with respect to voltage applied. Four different hole sizes of 5 μm, 2μm, 1μm, and 500 nm are patterned onto an electrode. At 5 V/cm, no deposition is seen in any of the holes. With increasing voltage, depositions into subsequently smaller-sized holes are observed. At 22.5 V/cm, a monolayer deposition occurs whereas at 30 V/cm, multilayered deposition occurs. The SEM scale bar is 2 μm.
See On-Demand and Location Selective Particle Assembly via Electrophoretic Deposition for Fabricating Structures with Particleto-Particle Precision, Fang Qian, Andrew J. Pascall, Mihail Bora, T. Yong-Jin Han, Shirui Guo, Sonny S. Ly, Marcus A. Worsley, Joshua D. Kuntz, and Tammy Y. Olson. Langmuir 2015, 31, 3563−3568. dx.doi.org/10.1021/la502724n
Contact: Tim Carpenter
Glutamate Receptors are key neuroreceptors in the brain that have been linked to a number of major neurodegenerative diseases. They are massive, multi-domain, membrane-embedded proteins. Their sheer size and complexity makes them an ideal subject for simulation using Lawrence Livermore National Laboratory's world-class supercomputers.
The ligand-induced closing actions of the ligand-binding domains of Glutamate Receptors determine the opening pathway of the receptor channel. By studying how these conformational pathways progress, we can investigate possible intermediate states that may exist, and probe potential target sites for drug design.
This image shows the overall structure of the Glutamate Receptor, with its "base" embedded in the neuronal cell membrane. The
ligand-binding domains are situated just above the membrane.
These two animations show two possible pathways for the ligand-binding domains to "close" around a ligand (which is not shown). The first pathway has the lower lobe smoothly moving up to contact the upper lobe, while the second pathway incorporates a slight twisting motion of the lower lobe as it closes. Ongoing research explores the relative energetics of such alternative conformational paths with molecular dynamics all-atom computer simulations.
Contact: Nicholas Fischer
a) Functionalizing nanolipoprotein particles
We are developing nanolipoprotein particles (NLPs) for myriad biotechnology applications. These biocompatible mimetics of high density lipoproteins (HDLs) are readily prepared from purified apolipoproteins and phospholipids. Due to the robust nature of NLP self-assembly, amphipathic molecules, including lipids bearing functional moieties at their head group, can be incorporated into the resultant lipid bilayer. In this manner, specific functionality can be imparted to the NLP itself, providing a means for biomolecule conjugation, fluorescent or contrast agent labeling, and drug or adjuvant incorporation.
b) Vaccine platform for subunit antigens
Developing vaccines using subunit antigens is a safe alternative to traditional vaccines, yet requires a combination of antigen delivery and additional stimulation of the immune system to provide protective efficacy. We have demonstrated that antigens and adjuvants can be co-localized on NLPs, and that these co-localized formulations perform far superior than the individually co-administered formulations.
c) Immunomodulation of host response against pathogens
Priming the host immune system to attack any invading pathogen is a broad-spectrum countermeasure that provides a means for immediate treatment in the event of nature or engineered outbreaks. Co-delivery of innate immune agonists may provide rapid immune stimulation while reducing unwanted side-effects. We are currently developing NLPs for the delivery of agonist combinations. The inherent NLP amphipathicity, as well as ease of functionalization, allow NLPs to readily accommodate agonists of disparate chemistries, including small molecules, oligonucleotides, and biomolecules.
See Colocalized delivery of adjuvant and antigen using nanolipoprotein particles enhances the immune response to recombinant antigens. Journal of the American Chemical Society, 135, 2044-2047 (2013).
Contact: Nick Fischer
DNA aptamers are short single-stranded DNA molecules that can be developed to bind their molecular targets with nanomolar affinity, and provide an alternative to antibodies in many cases. The identification of DNA sequences that can bind a particular target requires the selection process (known as SELEX) that ultimately generates at least 100 potential DNA molecules that display affinity for that target. The characterization of these down-selected sequences is the greatest current bottleneck in this process. We have developed DNA aptamer arrays that can be used to interrogate tens of thousands of DNA strands simultaneously. This is an efficient approach for reducing these DNA strands to their minimal binding region as well as providing significant information on aptamer sequence, structure, and function. We have also developed approaches for sensitive biomolecule detection based on DNA amplification techniques, including PCR and rolling-circle amplification.
Contact: Yongqin Jiao
The aerobic, aquatic, freshwater bacterium Caulobacter crescentus has been shown to be highly tolerant of the water soluble form of Uranium, U(VI). U(VI) poses a signficant threat to human health and wildlife as an environmental contaminant. Our aim is to understand Uranium detoxification and biomineralization
processes in C. crescentus.
Phosphate Metabolism Facilitates Cell Survival under Uranium Stress. Caulobacter crescentus produces biomolecules that can serve as templates for the formation and growth of uranium minerals, facilitating uranium biomineralization. In particular, phophatase enzymes -- both cell-bound and in-solution -- aid the precipitation of uranium phosphate minerals that form extracellularly. (In the transmission electron micrograph, uranium minerals are the bright areas.) Poster
Contact: Tim Carpenter
Neurotransmitters convey signals from one neuron to the next and are indispensable to the functioning of the nervous system. These small molecules bind to receptors to exert their action. One of the most important neurotransmitters is gamma-aminobutyric acid (GABA), which binds to its type A receptor (GABAA-receptor) to exert an inhibitory influence on the neuron. Many drugs, both medicinal and nefarious, bind to these GABAA-receptors and alter the balance of neuronal signals in the brain. There is a fine balance between these drugs eliciting the desired effect, and causing unwanted and sometimes irreversible alterations in neural behavior.
The average GABA binding pathway illustrates the narrowing of the standard deviation of GABA molecule positions as the molecule approaches the binding site. Each disk is centered at the average GABA position at that distance from the binding site (red circle). The disk's diameter is proportional to the standard deviation of the GABA position at that distance. The disks are oriented with their axes intersecting the binding site. The ligand-binding domain of the receptor is shown in gray.
To study this critical binding event, we used many molecular dynamics computational simulations to observe precisely how the GABA molecule binds to GABAA-receptor. One hundred individual simulations were carried out where GABA was placed near the binding site and then allowed to freely bind to the GABAA-receptor. Binding occurred in 19 of these simulations. Statistical analysis of these binding simulations reveals the consistent electrostatics-driven pathway taken by GABA molecules to enter the binding site. Improved understanding of binding events enables the development of safer medicinal neuroactive drugs and countermeasures for effects of neuronal chemical trauma.
See An Electrostatic Funnel in the GABA-Binding Pathway Timothy S. Carpenter and Felice C. Lightstone PLoS Comput. Biol. (2016) 12(4): e1004831.
The average dipole experienced by GABA molecules as they follow the binding pathway indicates that the molecules are aligned in the protein’s electrostatic field. The orientation of the average dipole of the GABA molecules at that distance from the reaction site (red circle) is represented by an arrow, with the length of the arrow proportional to the strength of the average dipole. Blue-gray-red "filaments" show selected electric field-lines.
Contact: Felice Lightstone
Figure 1. The initial fragment hit, bound to E. faecalis GyrB. The coloring of the GyrB enzyme indicates residue conservation across members of the GyrB evolutionary family; green regions are identical across members, yellow are conserved regions, and red are variable/non-conserved regions.
The increasing spread of bacterial strains that are resistant to current antibiotic drugs is a serious problem. Structure-based drug design using high-performance computing has helped create the first new class of antibiotics in 30 years. This promising drug candidate has broad activity and effectiveness, and in addition, will hamper the evolution of resistance among its bacterial targets.
A number of important antibiotics target the bacterial topoisomerases DNA gyrase (GyrB) or topoisomerase IV (ParE), which are essential enzymes that control the topological state of DNA during replication. The effectiveness of these drugs, however, is being lost due to the spread of drug-resistant bacterial strains. While disabling either GyrB or ParE will kill the bacterium, a “dual-target” drug, which disables both enzymes, is less likely to succumb to drug resistance because two independent advantageous mutations would have to occur simultaneously in a bacterium in order for it to survive.
Figure 2. Chemical structure of initial fragment.
Figure 3. Scaffold of the molecule, with potential R groups labeled.
Development of the candidate antibiotic began with the identification of a molecular fragment that weakly disrupts the activity of both the GyrB and ParE enzymes through its interaction with GyrB and ParE inhibitory binding pockets. The fragment binds to the pocket as Figure 1 shows with GyrB from E. faecalis.
The scaffold of this fragment has several R groups that can be modified or added; Figure 2 shows the chemical structure of the fragment, and Figure 3 shows the scaffold with R groups labeled. The R groups interact with the GyrB binding pocket in different ways, as Figure 4 shows.
Figure 4. View of the binding pocket surface. The potential hydrogen-bonds between the molecule and conserved adenine-binding aspartate and structural water molecule are shown. The R5 and R6 groups face the active-site pocket interior, while the R2 and R4 groups are directed towards partially solvent-exposed faces of the active-site pocket.
Potential R-group modifications were first explored with virtual screening, computational molecular dynamics simulations, and free-energy calculations, as well as quantum mechanical calculations, of the candidate drug molecules. Modifications that would produce increased affinity of the drug to GyrB and ParE were identified; the best of these were synthesized and tested in vitro.
High-performance computing enables the quick turnaround of high-quality, compute-intensive calculations. This was important because of the short development cycle – test, modify, test – involved in this effort.
Many, many alternatives were considered. Timely computer-based screening of options helps focus development efforts and helps to minimize costly wet-lab expenditures, such as drug synthesis.
Alternatives explored included substituents in three distinct regions of the active-site pocket: (i) the lipophilic active-site interior, along the R5 and R6 group vectors, (ii) the salt-bridge pocket, along the R2 group vector, and (iii) the residues and ordered solvent network at the mouth of the lipophilic pocket, along the R4 group vector. Filling the lipophilic pocket interior via installation of an ethyl group at R6 and a small substituent at R5 resulted in drug molecules with potencies two to three orders of magnitude greater than that of the initial fragment.
Particular attention was paid to optimizing the dual-targeting capabilities of the drug molecule, that is, affinity for both GyrB and ParE, while also minimizing potential off-target activity.
Thus, this drug development effort used high-performance computing to explore structure-based inhibitor design against multiple members of the target protein family, resulting in an inhibitor series with exquisite potency, broad enzymatic spectrum, and dual-targeting activity. The series is highly ligand-efficient, and tolerates significant chemical diversity at two R-group locations without compromising enzyme potency and spectrum. Taken together, these features greatly improve the prospects for developing molecules with antibacterial activity and good drug-like properties.
This work was done in collaboration with Trius Therapeutics, Inc., San Diego, CA
See L. W. Tari, X. Li, M. Trzoss, D. C. Bensen, Z. Chen, T. Lam, J. Zhang, S. J. Lee, G. Hough, D. Phillipson, S. Akers-Rodriguez, M. L. Cunningham, B. P. Kwan, K. J. Nelson, A. Castellano, J. B. Locke, V. Brown-Driver, T. M. Murphy, V. S. Ong, C. M. Pillar, D. L. Shinabarger, J. Nix, F. C. Lightstone, S. E. Wong, T. B. Nguyen, K. J. Shaw, J. Finn (2013) Tricyclic GyrB/ParE (TriBE) Inhibitors: A New Class of Broad-Spectrum Dual-Targeting Antibacterial Agents. PLoS ONE, 8(12): e84409.