Matching Items (274)
Description
Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the room temperature crystal structure of the chromophore-binding domains of the Deinococcus radiodurans phytochrome at 2.1 Å resolution. The structure was obtained by serial femtosecond X-ray crystallography from microcrystals at an X-ray free electron laser. We find overall good agreement compared to a crystal structure at 1.35 Å resolution derived from conventional crystallography at cryogenic temperatures, which we also report here. The thioether linkage between chromophore and protein is subject to positional ambiguity at the synchrotron, but is fully resolved with SFX. The study paves the way for time-resolved structural investigations of the phytochrome photocycle with time-resolved SFX.
ContributorsEdlund, Petra (Author) / Takala, Heikki (Author) / Claesson, Elin (Author) / Henry, Leocadie (Author) / Dods, Robert (Author) / Lehtivuori, Heli (Author) / Panman, Matthijs (Author) / Pande, Kanupriya (Author) / White, Thomas (Author) / Nakane, Takanori (Author) / Berntsson, Oskar (Author) / Gustavsson, Emil (Author) / Bath, Petra (Author) / Modi, Vaibhav (Author) / Roy Chowdhury, Shatabdi (Author) / Zook, James (Author) / Berntsen, Peter (Author) / Pandey, Suraj (Author) / Poudyal, Ishwor (Author) / Tenboer, Jason (Author) / Kupitz, Christopher (Author) / Barty, Anton (Author) / Fromme, Petra (Author) / Koralek, Jake D. (Author) / Tanaka, Tomoyuki (Author) / Spence, John (Author) / Liang, Mengning (Author) / Hunter, Mark S. (Author) / Boutet, Sebastien (Author) / Nango, Eriko (Author) / Moffat, Keith (Author) / Groenhof, Gerrit (Author) / Ihalainen, Janne (Author) / Stojkovic, Emina A. (Author) / Schmidt, Marius (Author) / Westenhoff, Sebastian (Author) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / Department of Physics (Contributor)
Created2016-10-19
Description
Antibodies are essential for structural determinations and functional studies of membrane proteins, but antibody generation is limited by the availability of properly-folded and purified antigen. We describe the first application of genetic immunization to a structurally diverse set of membrane proteins to show that immunization of mice with DNA alone produced antibodies against 71% (n = 17) of the bacterial and viral targets. Antibody production correlated with prior reports of target immunogenicity in host organisms, underscoring the efficiency of this DNA-gold micronanoplex approach. To generate each antigen for antibody characterization, we also developed a simple in vitro membrane protein expression and capture method. Antibody specificity was demonstrated upon identifying, for the first time, membrane-directed heterologous expression of the native sequences of the FopA and FTT1525 virulence determinants from the select agent Francisella tularensis SCHU S4. These approaches will accelerate future structural and functional investigations of therapeutically-relevant membrane proteins.
ContributorsHansen, Debra (Author) / Robida, Mark (Author) / Craciunescu, Felicia (Author) / Loskutov, Andrey (Author) / Dorner, Katerina (Author) / Rodenberry, John-Charles (Author) / Wang, Xiao (Author) / Olson, Tien (Author) / Patel, Hetal (Author) / Fromme, Petra (Author) / Sykes, Kathryn (Author) / Biodesign Institute (Contributor) / Innovations in Medicine (Contributor) / Applied Structural Discovery (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor)
Created2016-02-24
Description
Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 Å resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1 ms yields difference Fourier maps compatible with the dark to M state transition of bR. Importantly, the method is very sample efficient and reduces sample consumption to about 1 mg per collected time point. Accumulation of M intermediate within the crystal lattice is confirmed by time-resolved visible absorption spectroscopy. This study provides an important step towards characterizing the complete photocycle dynamics of retinal proteins and demonstrates the feasibility of a sample efficient viscous medium jet for TR-SFX.
ContributorsNogly, Przemyslaw (Author) / Panneels, Valerie (Author) / Nelson, Garrett (Author) / Gati, Cornelius (Author) / Kimura, Tetsunari (Author) / Milne, Christopher (Author) / Milathianaki, Despina (Author) / Kubo, Minoru (Author) / Wu, Wenting (Author) / Conrad, Chelsie (Author) / Coe, Jesse (Author) / Bean, Richard (Author) / Zhao, Yun (Author) / Bath, Petra (Author) / Dods, Robert (Author) / Harimoorthy, Rajiv (Author) / Beyerlein, Kenneth R. (Author) / Rheinberger, Jan (Author) / James, Daniel (Author) / Deponte, Daniel (Author) / Li, Chufeng (Author) / Sala, Leonardo (Author) / Williams, Garth J. (Author) / Hunter, Mark S. (Author) / Koglin, Jason E. (Author) / Berntsen, Peter (Author) / Nango, Eriko (Author) / Iwata, So (Author) / Chapman, Henry N. (Author) / Fromme, Petra (Author) / Frank, Matthias (Author) / Abela, Rafael (Author) / Boutet, Sebastien (Author) / Barty, Anton (Author) / White, Thomas A. (Author) / Weierstall, Uwe (Author) / Spence, John (Author) / Neutze, Richard (Author) / Schertler, Gebhard (Author) / Standfuss, Jorg (Author) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor) / Department of Chemistry and Biochemistry (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / School of Molecular Sciences (Contributor)
Created2016-08-22
Description
To achieve nitrite accumulation for shortcut biological nitrogen removal (SBNR) in a biofilm process, we explored the simultaneous effects of oxygen limitation and free ammonia (FA) and free nitrous acid (FNA) inhibition in the nitrifying biofilm. We used the multi-species nitrifying biofilm model (MSNBM) to identify conditions that should or should not lead to nitrite accumulation, and evaluated the effectiveness of those conditions with experiments in continuous flow biofilm reactors (CFBRs). CFBR experiments were organized into four sets with these expected outcomes based on the MSNBM as follows: (i) Control, giving full nitrification; (ii) oxygen limitation, giving modest long-term nitrite build up; (iii) FA inhibition, giving no long-term nitrite accumulation; and (iv) FA inhibition plus oxygen limitation, giving major long-term nitrite accumulation. Consistent with MSNBM predictions, the experimental results showed that nitrite accumulated in sets 2–4 in the short term, but long-term nitrite accumulation was maintained only in sets 2 and 4, which involved oxygen limitation. Furthermore, nitrite accumulation was substantially greater in set 4, which also included FA inhibition. However, FA inhibition (and accompanying FNA inhibition) alone in set 3 did not maintained long-term nitrite accumulation. Nitrite-oxidizing bacteria (NOB) activity batch tests confirmed that little NOB or only a small fraction of NOB were present in the biofilms for sets 4 and 2, respectively. The experimental data supported the previous modeling results that nitrite accumulation could be achieved with a lower ammonium concentration than had been required for a suspended-growth process. Additional findings were that the biofilm exposed to low dissolved oxygen (DO) limitation and FA inhibition was substantially denser and probably had a lower detachment rate.
ContributorsPark, Seongjun (Author) / Chung, Jinwook (Author) / Rittmann, Bruce (Author) / Bae, Wookeun (Author) / Biodesign Institute (Contributor) / Swette Center for Environmental Biotechnology (Contributor)
Created2015-01-01
Description
Chloroform and methanol are superior solvents for lipid extraction from photosynthetic microorganisms, because they can overcome the resistance offered by the cell walls and membranes, but they are too toxic and expensive to use for large-scale fuel production. Biomass from the photosynthetic microalga Scenedesmus, subjected to a commercially available pre-treatment technology called Focused-Pulsed® (FP), yielded 3.1-fold more crude lipid and fatty acid methyl ester (FAME) after extraction with a range of solvents. FP treatment increased the FAME-to-crude-lipid ratio for all solvents, which means that the extraction of non-lipid materials was minimized, while the FAME profile itself was unchanged compared to the control. FP treatment also made it possible to use only a small proportion of chloroform and methanol, along with isopropanol, to obtain equivalent yields of lipid and FAME as with 100% chloroform plus methanol.
ContributorsLai, Yenjung Sean (Author) / Parameswaran, Prathap (Author) / Li, Ang (Author) / Baez, Maria (Author) / Rittmann, Bruce (Author) / Biodesign Institute (Contributor) / Swette Center for Environmental Biotechnology (Contributor)
Created2014-12-01
Description
Sulfadiazine (SD), one of broad-spectrum antibiotics, exhibits limited biodegradation in wastewater treatment due to its chemical structure, which requires initial mono-oxygenation reactions to initiate its biodegradation. Intimately coupling UV photolysis with biodegradation, realized with the internal loop photobiodegradation reactor, accelerated SD biodegradation and mineralization by 35 and 71 %, respectively. The main organic products from photolysis were 2-aminopyrimidine (2-AP), p-aminobenzenesulfonic acid (ABS), and aniline (An), and an SD-photolysis pathway could be identified using C, N, and S balances. Adding An or ABS (but not 2-AP) into the SD solution during biodegradation experiments (no UV photolysis) gave SD removal and mineralization rates similar to intimately coupled photolysis and biodegradation. An SD biodegradation pathway, based on a diverse set of the experimental results, explains how the mineralization of ABS and An (but not 2-AP) provided internal electron carriers that accelerated the initial mono-oxygenation reactions of SD biodegradation. Thus, multiple lines of evidence support that the mechanism by which intimately coupled photolysis and biodegradation accelerated SD removal and mineralization was through producing co-substrates whose oxidation produced electron equivalents that stimulated the initial mono-oxygenation reactions for SD biodegradation.
ContributorsPan, Shihui (Author) / Yan, Ning (Author) / Liu, Xinyue (Author) / Wang, Wenbing (Author) / Zhang, Yongming (Author) / Liu, Rui (Author) / Rittmann, Bruce (Author) / Biodesign Institute (Contributor) / Swette Center for Environmental Biotechnology (Contributor)
Created2014-11-01
Description
Heterochromatin is a repressive chromatin compartment essential for maintaining genomic integrity. A hallmark of heterochromatin is the presence of specialized nonhistone proteins that alter chromatin structure to inhibit transcription and recombination. It is generally assumed that heterochromatin is highly condensed. However, surprisingly little is known about the structure of heterochromatin or its dynamics in solution. In budding yeast, formation of heterochromatin at telomeres and the homothallic silent mating type loci require the Sir3 protein. Here, we use a combination of sedimentation velocity, atomic force microscopy and nucleosomal array capture to characterize the stoichiometry and conformation of Sir3 nucleosomal arrays. The results indicate that Sir3 interacts with nucleosomal arrays with a stoichiometry of two Sir3 monomers per nucleosome. We also find that Sir3 fibres are less compact than canonical magnesium-induced 30 nm fibres. We suggest that heterochromatin proteins promote silencing by ‘coating’ nucleosomal arrays, stabilizing interactions between nucleosomal histones and DNA.
ContributorsSwygert, Sarah G. (Author) / Manning, Benjamin J. (Author) / Senapati, Subhadip (Author) / Kaur, Parminder (Author) / Lindsay, Stuart (Author) / Demeler, Borries (Author) / Peterson, Craig L. (Author) / Biodesign Institute (Contributor) / Single Molecule Biophysics (Contributor)
Created2014-08-01
Description
We studied the microbial community structure of pilot two-stage membrane biofilm reactors (MBfRs) designed to reduce nitrate (NO[subscript 3]–) and perchlorate (ClO[subscript 4]–) in contaminated groundwater. The groundwater also contained oxygen (O[subscript 2]) and sulfate (SO[2 over 4]–), which became important electron sinks that affected the NO[subscript 3]– and ClO[subscript 4]– removal rates. Using pyrosequencing, we elucidated how important phylotypes of each “primary” microbial group, i.e., denitrifying bacteria (DB), perchlorate-reducing bacteria (PRB), and sulfate-reducing bacteria (SRB), responded to changes in electron-acceptor loading. UniFrac, principal coordinate analysis (PCoA), and diversity analyses documented that the microbial community of biofilms sampled when the MBfRs had a high acceptor loading were phylogenetically distant from and less diverse than the microbial community of biofilm samples with lower acceptor loadings. Diminished acceptor loading led to SO[2 over 4]– reduction in the lag MBfR, which allowed Desulfovibrionales (an SRB) and Thiothrichales (sulfur-oxidizers) to thrive through S cycling. As a result of this cooperative relationship, they competed effectively with DB/PRB phylotypes such as Xanthomonadales and Rhodobacterales. Thus, pyrosequencing illustrated that while DB, PRB, and SRB responded predictably to changes in acceptor loading, a decrease in total acceptor loading led to important shifts within the “primary” groups, the onset of other members (e.g., Thiothrichales), and overall greater diversity.
ContributorsOntiveros-Valencia, Aura (Author) / Tang, Youneng (Author) / Zhao, Heping (Author) / Friese, David (Author) / Overstreet, Ryan (Author) / Smith, Jennifer (Author) / Evans, Patrick (Author) / Rittmann, Bruce (Author) / Krajmalnik-Brown, Rosa (Author) / Biodesign Institute (Contributor) / Swette Center for Environmental Biotechnology (Contributor) / Julie Ann Wrigley Global Institute of Sustainability (Contributor) / School of Sustainability (Contributor)
Created2014-07-01
Description
The Combined Activated Sludge-Anaerobic Digestion Model (CASADM) quantifies the effects of recycling anaerobic-digester (AD) sludge on the performance of a hybrid activated sludge (AS)-AD system. The model includes nitrification, denitrification, hydrolysis, fermentation, methanogenesis, and production/utilization of soluble microbial products and extracellular polymeric substances (EPS). A CASADM example shows that, while effluent COD and N are not changed much by hybrid operation, the hybrid system gives increased methane production in the AD and decreased sludge wasting, both caused mainly by a negative actual solids retention time in the hybrid AD. Increased retention of biomass and EPS allows for more hydrolysis and conversion to methane in the hybrid AD. However, fermenters and methanogens survive in the AS, allowing significant methane production in the settler and thickener of both systems, and AD sludge recycle makes methane formation greater in the hybrid system.
ContributorsYoung, Michelle (Author) / Marcus, Andrew (Author) / Rittmann, Bruce (Author) / Biodesign Institute (Contributor) / Swette Center for Environmental Biotechnology (Contributor)
Created2013-08-13
Description
Plant viral vectors have previously been used to produce high expression levels of antibodies and other proteins of interest. By utilizing a transformed Agrobacterium with the vector containing the protein of interest for infiltration, viral vectors can easily reach the plant cells making it an effective form of transient protein expression. For this project two different plant viral vectors were compared; the geminiviral vector derived from Bean yellow dwarf virus (BeYDV) and the MagnICON vector system derived from Tobacco Mosaic Virus(TMV) and Potato Virus X(PVX). E16, an antibody against West Nile virus, has previously been expressed using both systems but expression levels between the systems were not directly compared. Agrobacterium tumefaciens EHA105 cells were transformed with both systems and expression levels of E16 were quantified using ELISAs. Results showed very low expression levels of E16 using the geminiviral vector indicating a need for further investigation into the clone used as previous studies reported much higher expression levels with the system.
ContributorsMurphy, Skylar (Author) / Chen, Qiang (Thesis director) / Jugler, Collin (Committee member) / College of Health Solutions (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05