Matching Items (16)
Description
The transmembrane subunit (gp41) of the envelope glycoprotein of HIV-1 associates noncovalently with the surface subunit (gp120) and together they play essential roles in viral mucosal transmission and infection of target cells. The membrane proximal region (MPR, residues 649-683) of gp41 is highly conserved and contains epitopes of broadly neutralizing antibodies. The transmembrane (TM) domain (residues 684-705) of gp41 not only anchors the envelope glycoprotein complex in the viral membrane but also dynamically affects the interactions of the MPR with the membrane. While high-resolution X-ray structures of some segments of the MPR were solved in the past, they represent the pre-fusion and post-fusion conformations, most of which could not react with the broadly neutralizing antibodies 2F5 and 4E10. Structural information on the TM domain of gp41 is scant and at low resolution.
This thesis describes the structural studies of MPR-TM (residues 649-705) of HIV-1 gp41 by X-ray crystallography. MPR-TM was fused with different fusion proteins to improve the membrane protein overexpression. The expression level of MPR-TM was improved by fusion to the C-terminus of the Mistic protein, yielding ∼1 mg of pure MPR-TM protein per liter cell culture. The fusion partner Mistic was removed for final crystallization. The isolated MPR-TM protein was biophysically characterized and is a monodisperse candidate for crystallization. However, no crystal with diffraction quality was obtained even after extensive crystallization screens. A novel construct was designed to overexpress MPR-TM as a maltose binding protein (MBP) fusion. About 60 mg of MBP/MPR-TM recombinant protein was obtained from 1 liter of cell culture. Crystals of MBP/MPR-TM recombinant protein could not be obtained when MBP and MPR-TM were separated by a 42 amino acid (aa)-long linker but were obtained after changing the linker to three alanine residues. The crystals diffracted to 2.5 Å after crystallization optimization. Further analysis of the diffraction data indicated that the crystals are twinned. The final structure demonstrated that MBP crystallized as a dimer of trimers, but the electron density did not extend beyond the linker region. We determined by SDS-PAGE and MALDI-TOF MS that the crystals contained MBP only. The MPR-TM of gp41 might be cleaved during or after the process of crystallization. Comparison of the MBP trimer reported here with published trimeric MBP fusion structures indicated that MBP might form such a trimeric conformation under the effect of MPR-TM.
This thesis describes the structural studies of MPR-TM (residues 649-705) of HIV-1 gp41 by X-ray crystallography. MPR-TM was fused with different fusion proteins to improve the membrane protein overexpression. The expression level of MPR-TM was improved by fusion to the C-terminus of the Mistic protein, yielding ∼1 mg of pure MPR-TM protein per liter cell culture. The fusion partner Mistic was removed for final crystallization. The isolated MPR-TM protein was biophysically characterized and is a monodisperse candidate for crystallization. However, no crystal with diffraction quality was obtained even after extensive crystallization screens. A novel construct was designed to overexpress MPR-TM as a maltose binding protein (MBP) fusion. About 60 mg of MBP/MPR-TM recombinant protein was obtained from 1 liter of cell culture. Crystals of MBP/MPR-TM recombinant protein could not be obtained when MBP and MPR-TM were separated by a 42 amino acid (aa)-long linker but were obtained after changing the linker to three alanine residues. The crystals diffracted to 2.5 Å after crystallization optimization. Further analysis of the diffraction data indicated that the crystals are twinned. The final structure demonstrated that MBP crystallized as a dimer of trimers, but the electron density did not extend beyond the linker region. We determined by SDS-PAGE and MALDI-TOF MS that the crystals contained MBP only. The MPR-TM of gp41 might be cleaved during or after the process of crystallization. Comparison of the MBP trimer reported here with published trimeric MBP fusion structures indicated that MBP might form such a trimeric conformation under the effect of MPR-TM.
ContributorsGong, Zhen (Author) / Fromme, Petra (Thesis advisor) / Mor, Tsafrir (Thesis advisor) / Ros, Alexandra (Committee member) / Redding, Kevin (Committee member) / Arizona State University (Publisher)
Created2014
Description
Acquisition of fluorescence via autocatalytic processes is unique to few proteins in the natural world. Fluorescent proteins (FPs) have been integral to live-cell imaging techniques for decades; however, mechanistic information is still emerging fifty years after the discovery of the original green fluorescent protein (GFP). Modification of the fluorescence properties of the proteins derived from GFP allows increased complexity of experiments and consequently, information content of the data acquired. The importance of arginine-96 in GFP has been widely discussed. It has been established as vital to the kinetics of chromophore maturation and to the overall fold of GFP before post-translational self-modification. Its value during chromophore maturation has been demonstrated by mutational studies and a hypothesis proposed for its catalytic function. A strategy is described herein to determine its pKa value via NMR to determine whether Arg96 possesses the chemical capacity to function as a general base during GFP chromophore biosynthesis. Förster resonance energy transfer (FRET) techniques commonly employ Enhanced Cyan Fluorescent Proteins (ECFPs) and their derivatives as donor fluorophores useful in real-time, live-cell imaging. These proteins have a tryptophan-derived chromophore that emits light in the blue region of the visible spectrum. Most ECFPs suffer from fluorescence instability, which, coupled with their low quantum yield, makes data analysis unreliable. The structural heterogeneity of these proteins also results in undesirable photophysical characteristics. Recently, mCerulean3, a ten amino acid mutant of ECFP, was introduced as an optimized FRET-donor protein (1). The amino acids changed include a mobile residue, Asp148, which has been mutated to a glycine in the new construct, and Thr65 near the chromophore has been mutated to a serine, the wild-type residue at this location. I have solved the x-ray crystal structure of mCerulean3 at low pH and find that the pH-dependent isomerization has been eliminated. The chromophore is in the trans-conformation previously observed in Cerulean at pH 8. The mutations that increase the quantum yield and improve fluorescence brightness result in a stable, bright donor fluorophore well-suited for use in quantitative microscopic imaging.
ContributorsWatkins, Jennifer L (Author) / Wachter, Rebekka M. (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Allen, James P. (Committee member) / Arizona State University (Publisher)
Created2012
Description
The evolution of photosynthesis caused the oxygen-rich atmosphere in which we thrive today. Although the reaction centers involved in oxygenic photosynthesis probably evolved from a protein like the reaction centers in modern anoxygenic photosynthesis, modern anoxygenic reaction centers are poorly understood. One such anaerobic reaction center is found in Heliobacterium modesticaldum. Here, the photosynthetic properties of H. modesticaldum are investigated, especially as they pertain to its unique photochemical reaction center.
The first part of this dissertation describes the optimization of the previously established protocol for the H. modesticaldum reaction center isolation. Subsequently, electron transfer is characterized by ultrafast spectroscopy; the primary electron acceptor, a chlorophyll a derivative, is reduced in ~25 ps, and forward electron transfer occurs directly to a 4Fe-4S cluster in ~650 ps without the requirement for a quinone intermediate. A 2.2-angstrom resolution X-ray crystal structure of the homodimeric heliobacterial reaction center is solved, which is the first ever homodimeric reaction center structure to be solved, and is discussed as it pertains to the structure-function relationship in energy and electron transfer. The structure has a transmembrane helix arrangement similar to that of Photosystem I, but differences in antenna and electron transfer cofactor positions explain variations in biophysical comparisons. The structure is then compared with other reaction centers to infer evolutionary hypotheses suggesting that the ancestor to all modern reaction centers could reduce mobile quinones, and that Photosystem I added lower energy cofactors to its electron transfer chain to avoid the formation of singlet oxygen.
In the second part of this dissertation, hydrogen production rates of H. modesticaldum are quantified in multiple conditions. Hydrogen production only occurs in cells grown without ammonia, and is further increased by removal of N2. These results are used to propose a scheme that summarizes the hydrogen-production metabolism of H. modesticaldum, in which electrons from pyruvate oxidation are shuttled through an electron transport pathway including the reaction center, ultimately reducing nitrogenase. In conjunction, electron microscopy images of H. modesticaldum are shown, which confirm that extended membrane systems are not exhibited by heliobacteria.
The first part of this dissertation describes the optimization of the previously established protocol for the H. modesticaldum reaction center isolation. Subsequently, electron transfer is characterized by ultrafast spectroscopy; the primary electron acceptor, a chlorophyll a derivative, is reduced in ~25 ps, and forward electron transfer occurs directly to a 4Fe-4S cluster in ~650 ps without the requirement for a quinone intermediate. A 2.2-angstrom resolution X-ray crystal structure of the homodimeric heliobacterial reaction center is solved, which is the first ever homodimeric reaction center structure to be solved, and is discussed as it pertains to the structure-function relationship in energy and electron transfer. The structure has a transmembrane helix arrangement similar to that of Photosystem I, but differences in antenna and electron transfer cofactor positions explain variations in biophysical comparisons. The structure is then compared with other reaction centers to infer evolutionary hypotheses suggesting that the ancestor to all modern reaction centers could reduce mobile quinones, and that Photosystem I added lower energy cofactors to its electron transfer chain to avoid the formation of singlet oxygen.
In the second part of this dissertation, hydrogen production rates of H. modesticaldum are quantified in multiple conditions. Hydrogen production only occurs in cells grown without ammonia, and is further increased by removal of N2. These results are used to propose a scheme that summarizes the hydrogen-production metabolism of H. modesticaldum, in which electrons from pyruvate oxidation are shuttled through an electron transport pathway including the reaction center, ultimately reducing nitrogenase. In conjunction, electron microscopy images of H. modesticaldum are shown, which confirm that extended membrane systems are not exhibited by heliobacteria.
ContributorsGisriel, Christopher J (Author) / Redding, Kevin E (Thesis advisor) / Jones, Anne K (Committee member) / Allen, James P. (Committee member) / Arizona State University (Publisher)
Created2017
Description
Over the last century, X-ray crystallography has been established as the most successful technique for unravelling the structure-function relationship in molecules. For integral membrane proteins, growing well-ordered large crystals is a challenge and hence, there is room for improving current methods of macromolecular crystallography and for exploring complimentary techniques. Since protein function is deeply associated with its structural dynamics, static position of atoms in a macromolecule are insufficient to unlock the mechanism.
The availability of X-ray free electron lasers presents an opportunity to study micron-sized crystals that could be triggered (using light, small molecules or physical conditions) to capture macromolecules in action. This method of ‘Time-resolved serial crystallography’ answers key biological questions by capturing snapshots of conformational changes associated with multi-step reactions. This dissertation describes approaches for studying structures of large membrane protein complexes. Both macro and micro-seeding techniques have been implemented for improving crystal quality and obtaining high-resolution structures. Well-diffracting 15-20 micron crystals of active Photosystem II were used to perform time-resolved studies with fixed-target Roadrunner sample delivery system. By employing continuous diffraction obtained up to 2 A, significant progress can be made towards understanding the process of water oxidation.
Structure of Photosystem I was solved to 2.3 A by X-ray crystallography and to medium resolution of 4.8 A using Cryogenic electron microscopy. Using complimentary techniques to study macromolecules provides an insight into differences among methods in structural biology. This helps in overcoming limitations of one specific technique and contributes in greater knowledge of the molecule under study.
The availability of X-ray free electron lasers presents an opportunity to study micron-sized crystals that could be triggered (using light, small molecules or physical conditions) to capture macromolecules in action. This method of ‘Time-resolved serial crystallography’ answers key biological questions by capturing snapshots of conformational changes associated with multi-step reactions. This dissertation describes approaches for studying structures of large membrane protein complexes. Both macro and micro-seeding techniques have been implemented for improving crystal quality and obtaining high-resolution structures. Well-diffracting 15-20 micron crystals of active Photosystem II were used to perform time-resolved studies with fixed-target Roadrunner sample delivery system. By employing continuous diffraction obtained up to 2 A, significant progress can be made towards understanding the process of water oxidation.
Structure of Photosystem I was solved to 2.3 A by X-ray crystallography and to medium resolution of 4.8 A using Cryogenic electron microscopy. Using complimentary techniques to study macromolecules provides an insight into differences among methods in structural biology. This helps in overcoming limitations of one specific technique and contributes in greater knowledge of the molecule under study.
ContributorsRoy Chowdhury, Shatabdi (Author) / Fromme, Petra (Thesis advisor) / Ros, Alexandra (Committee member) / Redding, Kevin (Committee member) / Arizona State University (Publisher)
Created2018
Description
CTB-MPR649-684 is a translational fusion protein consisting of the cholera toxin B subunit (CTB) and the conserved residues 649-684 of gp41 membrane proximal region (MPR). It is a candidate vaccine component aimed at early steps of the HIV-1 infection by blocking viral mucosal transmission. Bacterially produced CTB-MPR was previously shown to induce HIV-1 transcytosis-blocking antibodies in mice and rabbits. However, the induction of high-titer MPR specific antibodies with HIV-1 transcytosis blocking ability remains a challenge as the immuno-dominance of CTB overshadows the response to MPR. X-ray crystallography was used to investigate the structure of CTB-MPR with the goal of identifying potential solutions to improve the immune response of MPR. Various CTB-MPR variants were designed using different linkers connecting the two fusion proteins. The procedures for over-expression E. coli and purification have been optimized for each of the variants of CTB-MPR. The purity and oligomeric homogeneity of the fusion protein was demonstrated by electrophoresis, size-exclusion chromatography, dynamic light scattering, and immuno-blot analysis. Crystallization conditions for macroscopic and micro
ano-crystals have been established for the different variants of the fusion protein. Diffraction patterns were collected by using both conventional and serial femto-second crystallography techniques. The two crystallography techniques showed very interesting differences in both the crystal packing and unit cell dimensions of the same CTB-MPR construct. Although information has been gathered on CTB-MPR, the intact structure of fusion protein was not solved as the MPR region showed only weak electron density or was cleaved during crystallization of macroscopic crystals. The MPR region is present in micro
ano-crystals, but due to the severe limitation of the Free Electron Laser beamtime, only a partial data set was obtained and is insufficient for structure determination. However, the work of this thesis has established methods to purify large quantities of CTB-MPR and has established procedures to grow crystals for X-ray structure analysis. This has set the foundation for future structure determination experiments as well as immunization studies.
ano-crystals have been established for the different variants of the fusion protein. Diffraction patterns were collected by using both conventional and serial femto-second crystallography techniques. The two crystallography techniques showed very interesting differences in both the crystal packing and unit cell dimensions of the same CTB-MPR construct. Although information has been gathered on CTB-MPR, the intact structure of fusion protein was not solved as the MPR region showed only weak electron density or was cleaved during crystallization of macroscopic crystals. The MPR region is present in micro
ano-crystals, but due to the severe limitation of the Free Electron Laser beamtime, only a partial data set was obtained and is insufficient for structure determination. However, the work of this thesis has established methods to purify large quantities of CTB-MPR and has established procedures to grow crystals for X-ray structure analysis. This has set the foundation for future structure determination experiments as well as immunization studies.
ContributorsLee, Ho-Hsien (Author) / Fromme, Petra (Thesis advisor) / Mor, Tsafrir (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2015
Description
X-ray crystallography is the most widely used method to determine the structure of proteins, providing an understanding of their functions in all aspects of life to advance applications in fields such as drug development and renewable energy. New techniques, namely serial femtosecond crystallography (SFX), have unlocked the ability to unravel the structures of complex proteins with vital biological functions. A key step and major bottleneck of structure determination is protein crystallization, which is very arduous due to the complexity of proteins and their natural environments. Furthermore, crystal characteristics govern data quality, thus need to be optimized to attain the most accurate reconstruction of the protein structure. Crystal size is one such characteristic in which narrowed distributions with a small modal size can significantly reduce the amount of protein needed for SFX. A novel microfluidic sorting platform was developed to isolate viable ~200 nm – ~600 nm photosystem I (PSI) membrane protein crystals from ~200 nm – ~20 μm crystal samples using dielectrophoresis, as confirmed by fluorescence microscopy, second-order nonlinear imaging of chiral crystals (SONICC), and dynamic light scattering. The platform was scaled-up to rapidly provide 100s of microliters of sorted crystals necessary for SFX, in which similar crystal size distributions were attained. Transmission electron microscopy was used to view the PSI crystal lattice, which remained well-ordered postsorting, and SFX diffraction data was obtained, confirming a high-quality, viable crystal sample. Simulations indicated sorted samples provided accurate, complete SFX datasets with 3500-fold less protein than unsorted samples. Microfluidic devices were also developed for versatile, rapid protein crystallization screening using nanovolumes of sample. Concentration gradients of protein and precipitant were generated to crystallize PSI, phycocyanin, and lysozyme using modified counterdiffusion. Additionally, a passive mixer was created to generate unique solution concentrations within isolated nanowells to crystallize phycocyanin and lysozyme. Crystal imaging with brightfield microscopy, UV fluorescence, and SONICC coupled with numerical modeling allowed quantification of crystal growth conditions for efficient phase diagram development. The developed microfluidic tools demonstrated the capability of improving samples for protein crystallography, offering a foundation for continued development of platforms to aid protein structure determination.
ContributorsAbdallah, Bahige G (Author) / Ros, Alexandra (Thesis advisor) / Buttry, Daniel (Committee member) / Hayes, Mark (Committee member) / Arizona State University (Publisher)
Created2016
Description
Phase problem has been long-standing in x-ray diffractive imaging. It is originated from the fact that only the amplitude of the scattered wave can be recorded by the detector, losing the phase information. The measurement of amplitude alone is insufficient to solve the structure. Therefore, phase retrieval is essential to structure determination with X-ray diffractive imaging. So far, many experimental as well as algorithmic approaches have been developed to address the phase problem. The experimental phasing methods, such as MAD, SAD etc, exploit the phase relation in vector space. They usually demand a lot of efforts to prepare the samples and require much more data. On the other hand, iterative phasing algorithms make use of the prior knowledge and various constraints in real and reciprocal space. In this thesis, new approaches to the problem of direct digital phasing of X-ray diffraction patterns from two-dimensional organic crystals were presented. The phase problem for Bragg diffraction from two-dimensional (2D) crystalline monolayer in transmission may be solved by imposing a compact support that sets the density to zero outside the monolayer. By iterating between the measured stucture factor magnitudes along reciprocal space rods (starting with random phases) and a density of the correct sign, the complex scattered amplitudes may be found (J. Struct Biol 144, 209 (2003)). However this one-dimensional support function fails to link the rod phases correctly unless a low-resolution real-space map is also available. Minimum prior information required for successful three-dimensional (3D) structure retrieval from a 2D crystal XFEL diffraction dataset were investigated, when using the HIO algorithm. This method provides an alternative way to phase 2D crystal dataset, with less dependence on the high quality model used in the molecular replacement method.
ContributorsZhao, Yun (Author) / Spence, John C.H. (Thesis advisor) / Schmidt, Kevin (Committee member) / Weierstall, Uwe (Committee member) / Kirian, Richard (Committee member) / Zatsepin, Nadia (Committee member) / Arizona State University (Publisher)
Created2016
Description
Serial crystallography (SX) is a relatively new structural biology technique that collects X-ray diffraction data from microcrystals via femtosecond pulses produced by an X-ray free electron laser (X-FEL) or by synchrotron radiation, allowing for challenging protein structures to be solved from microcrystals at room temperature. Because of the youth of this technique, method development is necessary for it to achieve its full potential.
Most serial crystallography experiments have relied on delivering sample in the mother liquor focused into a stream by compressed gas. This liquid stream moves at a fast rate, meaning that most of the valuable sample is wasted. For this reason, the liquid jet can require 10-100 milligrams of sample for a complete data set. Agarose has been developed as a slow moving microcrystal carrier to decrease sample consumption and waste. The agarose jet provides low background, no Debye-Sherrer rings, is compatible for sample delivery in vacuum environments, and is compatible with a wide variety of crystal systems. Additionally, poly(ethylene oxide) which is amenable for data collection in atmosphere has been developed for synchrotron experiments. Thus this work allows sample limited proteins of difficult to crystallize systems to be investigated by serial crystallography.
Time-resolved serial X-ray crystallography (TR-SX) studies have only been employed to study light-triggered reactions in photoactive systems. While these systems are very important, most proteins in Nature are not light-driven. However, fast mixing of two liquids, such as those containing enzyme protein crystals and substrates, immediately before being exposed to an X-ray beam would allow conformational changes and /or intermediates to be seen by diffraction. As a model, 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS), has been developed for TR-SX. This enzyme initializes the first step of lipopolysaccharide synthesis by a net aldol condensation between arabinose-5-phosphate, phosphoenol pyruvate, and water. During this reaction, a short lived intermediate is formed and has been observed on a millisecond timescale using other methods. Thus KDO8PS is an ideal model protein for studying diffusion times into a crystal and short mixing times (<10 ms). For these experiments, microcrystals diffracting to high resolution have been developed and characterized.
Most serial crystallography experiments have relied on delivering sample in the mother liquor focused into a stream by compressed gas. This liquid stream moves at a fast rate, meaning that most of the valuable sample is wasted. For this reason, the liquid jet can require 10-100 milligrams of sample for a complete data set. Agarose has been developed as a slow moving microcrystal carrier to decrease sample consumption and waste. The agarose jet provides low background, no Debye-Sherrer rings, is compatible for sample delivery in vacuum environments, and is compatible with a wide variety of crystal systems. Additionally, poly(ethylene oxide) which is amenable for data collection in atmosphere has been developed for synchrotron experiments. Thus this work allows sample limited proteins of difficult to crystallize systems to be investigated by serial crystallography.
Time-resolved serial X-ray crystallography (TR-SX) studies have only been employed to study light-triggered reactions in photoactive systems. While these systems are very important, most proteins in Nature are not light-driven. However, fast mixing of two liquids, such as those containing enzyme protein crystals and substrates, immediately before being exposed to an X-ray beam would allow conformational changes and /or intermediates to be seen by diffraction. As a model, 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS), has been developed for TR-SX. This enzyme initializes the first step of lipopolysaccharide synthesis by a net aldol condensation between arabinose-5-phosphate, phosphoenol pyruvate, and water. During this reaction, a short lived intermediate is formed and has been observed on a millisecond timescale using other methods. Thus KDO8PS is an ideal model protein for studying diffusion times into a crystal and short mixing times (<10 ms). For these experiments, microcrystals diffracting to high resolution have been developed and characterized.
ContributorsConrad, Chelsie E (Author) / Fromme, Petra (Thesis advisor) / Ros, Alexandra (Committee member) / Allen, James (Committee member) / Arizona State University (Publisher)
Created2016
Description
The majority of trust research has focused on the benefits trust can have for individual actors, institutions, and organizations. This “optimistic bias” is particularly evident in work focused on institutional trust, where concepts such as procedural justice, shared values, and moral responsibility have gained prominence. But trust in institutions may not be exclusively good. We reveal implications for the “dark side” of institutional trust by reviewing relevant theories and empirical research that can contribute to a more holistic understanding. We frame our discussion by suggesting there may be a “Goldilocks principle” of institutional trust, where trust that is too low (typically the focus) or too high (not usually considered by trust researchers) may be problematic. The chapter focuses on the issue of too-high trust and processes through which such too-high trust might emerge. Specifically, excessive trust might result from external, internal, and intersecting external-internal processes. External processes refer to the actions institutions take that affect public trust, while internal processes refer to intrapersonal factors affecting a trustor’s level of trust. We describe how the beneficial psychological and behavioral outcomes of trust can be mitigated or circumvented through these processes and highlight the implications of a “darkest” side of trust when they intersect. We draw upon research on organizations and legal, governmental, and political systems to demonstrate the dark side of trust in different contexts. The conclusion outlines directions for future research and encourages researchers to consider the ethical nuances of studying how to increase institutional trust.
ContributorsNeal, Tess M.S. (Author) / Shockley, Ellie (Author) / Schilke, Oliver (Author)
Created2016
Description
Rosalind Elsie Franklin worked with X-ray crystallography at King's College London, UK, and she helped determine the helical structure of DNA in the early 1950s. Franklin's research helped establish molecular genetics, a field that investigates how heredity works on the molecular level. The discovery of the structure of DNA also made future research possible into the molecular basis of embryonic development, genetic disorders, and gene manipulation.
ContributorsO'Connell, Lindsey (Author) / Drago, Mary (Editor) / Arizona State University. School of Life Sciences. Center for Biology and Society. Embryo Project Encyclopedia. (Publisher) / Arizona Board of Regents (Publisher)
Created2013-11-17