Matching Items (45)
Description
Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.
ContributorsLi, Dianfan (Author) / Stansfeld, Phillip J. (Author) / Sansom, Mark S. P. (Author) / Keogh, Aaron (Author) / Vogeley, Lutz (Author) / Howe, Nicole (Author) / Lyons, Joseph A. (Author) / Aragao, David (Author) / Fromme, Petra (Author) / Fromme, Raimund (Author) / Basu, Shibom (Author) / Grotjohann, Ingo (Author) / Kupitz, Christopher (Author) / Rendek, Kimberley (Author) / Weierstall, Uwe (Author) / Zatsepin, Nadia (Author) / Cherezov, Vadim (Author) / Liu, Wei (Author) / Bandaru, Sateesh (Author) / English, Niall J. (Author) / Gati, Cornelius (Author) / Barty, Anton (Author) / Yefanov, Oleksandr (Author) / Chapman, Henry N. (Author) / Diederichs, Kay (Author) / Messerschmidt, Marc (Author) / Boutet, Sebastien (Author) / Williams, Garth J. (Author) / Seibert, M. Marvin (Author) / Caffrey, Martin (Author) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / Department of Physics (Contributor)
Created2015-12-17
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
G protein-coupled receptors, or GPCRs, are receptors located within the membrane of cells that elicit a wide array of cellular responses through their interactions with G proteins. Recent advances in the use of lipid cubic phase (LCP) for the crystallization of GPCRs, as well as increased knowledge of techniques to improve receptor stability, have led to a large increase in the number of available GPCR structures, despite historic difficulties. This project is focused on the histamine family of receptors, which are Class A GPCRs that are involved in the body’s allergic and inflammatory responses. In particular, the goal of this project was to design, express, and purify histamine receptors with the ultimate goal of crystallization. Successive rounds of optimization included the use of recombinant DNA techniques in E.coli to truncate sections of the proteins and the insertion of several fusion partner proteins to improve receptor expression and stability. All constructs were expressed in a Bac-to-Bac baculovirus expression system using Sf9 insect cells, solubilized using n-Dodecyl-β-D-Maltoside (DDM), and purified using immobilized metal affinity chromatography. Constructs were then analyzed by SDS-Page, Western blot, and size-exclusion chromatography to determine their presence, purity, and homogeneity. Along with their expression data from insect cells, the most stable and homogeneous construct from each round was used to design successive optimizations. After 3 rounds of construct design for each receptor, much work remains to produce a stable sample that has the potential to crystallize. Future work includes further optimization of the insertion site of the fusion proteins, ligand screening for co-crystallization, optimization of purification conditions, and screening of potential thermostabilizing point mutations. Success in solving a structure will allow for a more detailed understanding of the receptor function in addition to its vital use in rational drug discovery.
ContributorsCosgrove, Steven Andrew (Author) / Liu, Wei (Thesis director) / Mills, Jeremy (Committee member) / Mazor, Yuval (Committee member) / W. P. Carey School of Business (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
The dopamine 2 receptor (D2R) is a Class A GPCR which is essential for signaling in the nervous system, and has been implicated in numerous illnesses. While there are over 50 currently approved drugs which act on D2R, the structure has never been determined in detail. Although crystallography has historically been difficult with GPCRs, in recent years many structures have been solved using lipidic cubic phase (LCP) crystallization techniques. Sample preparation for LCP crystallization typically requires optimization of genetic constructs, recombinant expression, and purification techniques in order to produce a sample with sufficient stability and homogeneity. This study compares several genetic constructs utilizing different promoters, fusion proteins, fusion positions, and truncations in order to determine a high quality construct for LCP crystallization of
D2R. All constructs were expressed using the Bac-to-bac baculovirus expression system, then extracted with n-Dodecyl-β-D-Maltoside (DDM) and purified using metal affinity chromatography. Samples were then tested for quantity, purity, and homogeneity using SDS-PAGE, western blot, and size-exclusion chromatography. High quality samples were chosen based on insect cell expression levels, purification yield, and stability estimated by the levels of homomeric protein relative to aggregated protein. A final construct was chosen with which to continue future studies in optimization of thermal stability and crystallization conditions. Future work on this project is required to produce a sample amenable to crystallization. Screening of ligands for co-crystallization,
thermostabilizing point mutations, and potentially optimization of extraction and purification techniques prior to crystallization trials. Solving the D2R structure will lead to an increased understanding of its signaling mechanism and the mechanisms of currently approved drugs, while also providing a basis for more effective structure-based drug design.
D2R. All constructs were expressed using the Bac-to-bac baculovirus expression system, then extracted with n-Dodecyl-β-D-Maltoside (DDM) and purified using metal affinity chromatography. Samples were then tested for quantity, purity, and homogeneity using SDS-PAGE, western blot, and size-exclusion chromatography. High quality samples were chosen based on insect cell expression levels, purification yield, and stability estimated by the levels of homomeric protein relative to aggregated protein. A final construct was chosen with which to continue future studies in optimization of thermal stability and crystallization conditions. Future work on this project is required to produce a sample amenable to crystallization. Screening of ligands for co-crystallization,
thermostabilizing point mutations, and potentially optimization of extraction and purification techniques prior to crystallization trials. Solving the D2R structure will lead to an increased understanding of its signaling mechanism and the mechanisms of currently approved drugs, while also providing a basis for more effective structure-based drug design.
ContributorsErler, Maya Marie (Author) / Liu, Wei (Thesis director) / He, Ximin (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Non-alcoholic fatty liver disease occurs when triglycerides are stored in the liver leading to irreversible scarring and damage of liver tissue. Inside the liver, adipose triglyceride lipase is responsible for the breaking down of triglycerides and is regulated by the inhibitor g0/g1 switch gene 2 (G0S2). G0S2 is proposed to be one of the targets against drug design for non-alcoholic fatty liver disease, and more information is needed on the structure of this protein to aid in drug discovery. Here I describe the expression of G0S2 in an E. coli system as well as purification and biophysical characterization of a functional G0S2 in amounts viable for solution state Nuclear Magnetic Resonance (NMR) spectroscopy. Initial spectra of the isotopically labeled protein show well dispersed 15N resonance lines, clean 13C resonances, and dominant a-helices characteristics. These results show that a prepared G0S2 construct is suitable for solution NMR such that 20 amino acids are now assigned in the G0S2 portion of the protein, allowing for further NMR work with this protein for structural discovery. Further work with a large oligomeric complex of G0S2 with Maltose Binding Protein also shows promise for future cryo-EM work.
ContributorsMoran, Michael William (Author) / Fromma, Petra (Thesis advisor) / Guo, Jia (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2020
Description
G protein-coupled receptors (GPCRs) are known to be modulated by membrane cholesterol levels, but whether or not the effects are caused by specific receptor-cholesterol interactions or cholesterol’s general effects on the membrane is not well-understood. Results from coarse-grained molecular dynamics (CGMD) simulations coupled and structural bioinformatics offer new insights into how cholesterol modulates GPCR function by showing cholesterol interactions with β2AR that agree with previously published data. Additionally, differential and specific cholesterol binding in the CCK receptor subfamily was observed while revealing a previously unreported Cholesterol Recognition Amino-acid Consensus (CRAC) sequence that is also conserved across 38% of class A GPCRs. Mutation of this conserved CRAC sequence of the β2AR affects cholesterol stabilization of the receptor in a lipid bilayer. Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) has proven highly successful for structure determination of challenging membrane proteins crystallized in lipidic cubic phase, however, as most techniques, it has limitations. Using an optimized SFX experimental setup in a helium atmosphere we determined the room temperature structure of the adenosine A2A receptor (A2AAR) at 2.0 Å resolution and compared it with previous A2AAR structures determined in vacuum and/or at cryogenic temperatures. Specifically, we demonstrated the capability of utilizing high XFEL beam transmissions, in conjunction with a high dynamic range detector, to collect high-resolution SFX data while reducing crystalline material consumption and shortening the collection time required for a complete data set.
The results of these studies provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases. Furthermore, the experimental setups presented herein can be applied to future molecular dynamics and SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize.
The results of these studies provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases. Furthermore, the experimental setups presented herein can be applied to future molecular dynamics and SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize.
ContributorsGeiger, James (Author) / Liu, Wei (Thesis advisor) / Mills, Jeremy (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2020
Description
This work advances structural and biophysical studies of three proteins important in disease. First protein of interest is the Francisella tularensis outer membrane protein A (FopA), which is a virulence determinant of tularemia. This work describes recombinant expression in Escherichia coli and successful purification of membrane translocated FopA. The purified protein was dimeric as shown by native polyacrylamide gel electrophoresis and small angle X-ray scattering (SAXS) analysis, with an abundance of β-strands based on circular dichroism spectroscopy. SAXS data supports the presence of a pore. Furthermore, protein crystals of membrane translocated FopA were obtained with preliminary X-ray diffraction data. The identified crystallization condition provides the means towards FopA structure determination; a valuable tool for structure-based design of anti-tularemia therapeutics.
Next, the nonstructural protein μNS of avian reoviruses was investigated using in vivo crystallization and serial femtosecond X-ray crystallography. Avian reoviruses infect poultry flocks causing significant economic losses. μNS is crucial in viral factory formation facilitating viral replication within host cells. Thus, structure-based targeting of μNS has the potential to disrupt intracellular viral propagation. Towards this goal, crystals of EGFP-tagged μNS (EGFP-μNS (448-605)) were produced in insect cells. The crystals diffracted to 4.5 Å at X-ray free electron lasers using viscous jets as crystal delivery methods and initial electron density maps were obtained. The resolution reported here is the highest described to date for μNS, which lays the foundation towards its structure determination.
Finally, structural, and functional studies of human Threonine aspartase 1 (Taspase1) were performed. Taspase1 is overexpressed in many liquid and solid malignancies. In the present study, using strategic circular permutations and X-ray crystallography, structure of catalytically active Taspase1 was resolved. The structure reveals the conformation of a 50 residues long fragment preceding the active side residue (Thr234), which has not been structurally characterized previously. This fragment adopted a straight helical conformation in contrast to previous predictions. Functional studies revealed that the long helix is essential for proteolytic activity in addition to the active site nucleophilic residue (Thr234) mediated proteolysis. Together, these findings enable a new approach for designing anti-cancer drugs by targeting the long helical fragment.
Next, the nonstructural protein μNS of avian reoviruses was investigated using in vivo crystallization and serial femtosecond X-ray crystallography. Avian reoviruses infect poultry flocks causing significant economic losses. μNS is crucial in viral factory formation facilitating viral replication within host cells. Thus, structure-based targeting of μNS has the potential to disrupt intracellular viral propagation. Towards this goal, crystals of EGFP-tagged μNS (EGFP-μNS (448-605)) were produced in insect cells. The crystals diffracted to 4.5 Å at X-ray free electron lasers using viscous jets as crystal delivery methods and initial electron density maps were obtained. The resolution reported here is the highest described to date for μNS, which lays the foundation towards its structure determination.
Finally, structural, and functional studies of human Threonine aspartase 1 (Taspase1) were performed. Taspase1 is overexpressed in many liquid and solid malignancies. In the present study, using strategic circular permutations and X-ray crystallography, structure of catalytically active Taspase1 was resolved. The structure reveals the conformation of a 50 residues long fragment preceding the active side residue (Thr234), which has not been structurally characterized previously. This fragment adopted a straight helical conformation in contrast to previous predictions. Functional studies revealed that the long helix is essential for proteolytic activity in addition to the active site nucleophilic residue (Thr234) mediated proteolysis. Together, these findings enable a new approach for designing anti-cancer drugs by targeting the long helical fragment.
ContributorsNagaratnam, Nirupa (Author) / Fromme, Petra (Thesis advisor) / Johnston, Stephen (Thesis advisor) / Van Horn, Wade (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2020
Description
This work comprises a cumulative effort to provide analysis of proteins relevant to understanding and treating human disease. This dissertation focuses on two main protein complexes: the structure of the Chimp adenovirus Y25 capsid assembly, as used in the SARS-CoV-2 vaccine, Vaxzveria, and the Dbl family RhoGEF (guanosine exchange factor) Syx and its associated small G protein, RhoA. The course of research was influenced heavily by the onset of the Covid-19 pandemic and associated lockdown, which pushed anyone with the means to do meaningful research to shift priorities towards addressing the greatest public health crisis since the 1918 flu pandemic.
Analysis of the Syx-RhoA complex for the purposes of structurally guided drug design was initially the focus of heavy optimization efforts to overcome the numerous challenges associated with expression, purification, and handling of this protein. By analyzing E. Coli derived protein new important knowledge was gained about this protein’s biophysical characteristics which contribute to its behavior and may inform drug design efforts. Expression in SF9 insect cells resulted in promising conditions for production of homogeneous and monodispersed protein. Homology modeling and molecular dynamics simulation of this protein support hypotheses about its interactions with both RhoA as well as regions of the cytoplasmic leaflet of the cell membrane.
Structural characterization of ChAdOx1, the adenoviral vector used in the AstraZeneca Covid-19 vaccine, Vaxzveria resulted in the highest resolution adenovirus structure ever solved (3.07Å). Subsequent biochemical analysis and computational simulations of PF4 with the ChAdOx1 capsid reveal interactions with important implications for vaccine induced thrombocytic throbocytopenia syndrome, a disorder observed in approximately 0.000024% of patients who receive Vaxzveria.
ContributorsBoyd, Ryan J (Author) / Fromme, Petra (Thesis advisor) / Chiu, Po-Lin (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2021