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- All Subjects: Biochemistry
- Creators: Fromme, Petra
- Member of: Theses and Dissertations
- Member of: Barrett, The Honors College Thesis/Creative Project Collection
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Description
Conformational changes in biomolecules often take place on longer timescales than are easily accessible with unbiased molecular dynamics simulations, necessitating the use of enhanced sampling techniques, such as adaptive umbrella sampling. In this technique, the conformational free energy is calculated in terms of a designated set of reaction coordinates. At the same time, estimates of this free energy are subtracted from the potential energy in order to remove free energy barriers and cause conformational changes to take place more rapidly. This dissertation presents applications of adaptive umbrella sampling to a variety of biomolecular systems. The first study investigated the effects of glycosylation in GalNAc2-MM1, an analog of glycosylated macrophage activating factor. It was found that glycosylation destabilizes the protein by increasing the solvent exposure of hydrophobic residues. The second study examined the role of bound calcium ions in promoting the isomerization of a cis peptide bond in the collagen-binding domain of Clostridium histolyticum collagenase. This study determined that the bound calcium ions reduced the barrier to the isomerization of this peptide bond as well as stabilizing the cis conformation thermodynamically, and identified some of the reasons for this. The third study represents the application of GAMUS (Gaussian mixture adaptive umbrella sampling) to on the conformational dynamics of the fluorescent dye Cy3 attached to the 5' end of DNA, and made predictions concerning the affinity of Cy3 for different base pairs, which were subsequently verified experimentally. Finally, the adaptive umbrella sampling method is extended to make use of the roll angle between adjacent base pairs as a reaction coordinate in order to examine the bending both of free DNA and of DNA bound to the archaeal protein Sac7d. It is found that when DNA bends significantly, cations from the surrounding solution congregate on the concave side, which increases the flexibility of the DNA by screening the repulsion between phosphate backbones. The flexibility of DNA on short length scales is compared to the worm-like chain model, and the contribution of cooperativity in DNA bending to protein-DNA binding is assessed.
ContributorsSpiriti, Justin Matthew (Author) / van der Vaart, Arjan (Thesis advisor) / Chizmeshya, Andrew (Thesis advisor) / Matyushov, Dmitry (Committee member) / Fromme, Petra (Committee member) / Arizona State University (Publisher)
Created2011
Description
Membrane protein structure is continuing to be a topic of interest across the scientific community. However, high resolution structural data of these proteins is difficult to obtain. The amino acid transport protein, Outer Envelope Protein, 16kDa (OEP16) is a transmembrane protein channel that allows the passive diffusion of amino acids across the outer chloroplast membrane, and is used as a model protein in order to establish methods that ultimately reveal structural details about membrane proteins using nuclear magnetic resonance (NMR) spectroscopy. Methods include recombinant expression of isotope enriched inclusion bodies, purification and reconstitution in detergent micelles, and pre-characterization techniques including circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), and high pressure liquid chromatography (HPLC). High resolution NMR spectroscopy was able to assign 99% of the amide backbone and the chemical shifts provided detailed secondary structure of OEP16 on a per residue basis using the software TALOS+. Relaxation studies explored the intramolecular dynamics of OEP16 and results strongly support the resonance assignments. Successful titration studies were able to locate residues important for amino acid binding for import into the chloroplast as well as provide information on how the transmembrane helices of OEP16 are packed together. For the first time there is experimental evidence that can assign the location of secondary structure in OEP16 and creates a foundation for a future three dimensional structure.
ContributorsZook, James Duncan (Author) / Fromme, Petra (Thesis advisor) / Chen, Julian (Committee member) / Wachter, Rebekka (Committee member) / Arizona State University (Publisher)
Created2012
Description
The heliobacterial reaction center (HbRC) is widely considered the simplest and most primitive photosynthetic reaction center (RC) still in existence. Despite the simplicity of the HbRC, many aspects of the electron transfer mechanism remain unknown or under debate. Improving our understanding of the structure and function of the HbRC is important in determining its role in the evolution of photosynthetic RCs. In this work, the function and properties of the iron-sulfur cluster FX and quinones of the HbRC were investigated, as these are the characteristic terminal electron acceptors used by Type-I and Type-II RCs, respectively. In Chapter 3, I develop a system to directly detect quinone double reduction activity using reverse-phase high pressure liquid chromatography (RP-HPLC), showing that Photosystem I (PSI) can reduce PQ to PQH2. In Chapter 4, I use RP-HPLC to characterize the HbRC, showing a surprisingly small antenna size and confirming the presence of menaquinone (MQ) in the isolated HbRC. The terminal electron acceptor FX was characterized spectroscopically and electrochemically in Chapter 5. I used three new systems to reduce FX in the HbRC, using EPR to confirm a S=3/2 ground-state for the reduced cluster. The midpoint potential of FX determined through thin film voltammetry was -372 mV, showing the cluster is much less reducing than previously expected. In Chapter 7, I show light-driven reduction of menaquinone in heliobacterial membrane samples using only mild chemical reductants. Finally, I discuss the evolutionary implications of these findings in Chapter 7.
ContributorsCowgill, John (Author) / Redding, Kevin (Thesis advisor) / Jones, Anne (Committee member) / Fromme, Petra (Committee member) / Arizona State University (Publisher)
Created2012
Description
Time-resolved serial femtosecond crystallography is an emerging method that allows for structural discovery to be performed on biomacromolecules during their dynamic trajectory through a reaction pathway after activation. This is performed by triggering a reaction on an ensemble of molecules in nano- or microcrystals and then using femtosecond X-ray laser pulses produced by an X-ray free electron laser to collect near-instantaneous data on the crystal. A full data set can be collected by merging a sufficient number of these patterns together and multiple data sets can be collected at different points along the reaction pathway by manipulating the delay time between reaction initiation and the probing X-rays. In this way, these ‘snapshot’ structures can be viewed in series to make a molecular movie, allowing for atomic visualization of a molecule in action and, thereby, a structural basis for the mechanism and function of a given biomacromolecule.
This dissertation presents results towards this end, including the successful implementations of the first diffusive mixing chemoactivated reactions and ultrafast dynamics in the femtosecond regime. The primary focus is on photosynthetic membrane proteins and enzymatic drug targets, in pursuit of strategies for sustainable energy and medical advancement by gaining understanding of the structure-function relationships evolved in nature. In particular, photosystem I, photosystem II, the complex of photosystem I and ferredoxin, and 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase are reported on, from purification and isolation, to crystallogenesis, to experimental design and data collection and subsequent interpretation of results and novel insights gained.
This dissertation presents results towards this end, including the successful implementations of the first diffusive mixing chemoactivated reactions and ultrafast dynamics in the femtosecond regime. The primary focus is on photosynthetic membrane proteins and enzymatic drug targets, in pursuit of strategies for sustainable energy and medical advancement by gaining understanding of the structure-function relationships evolved in nature. In particular, photosystem I, photosystem II, the complex of photosystem I and ferredoxin, and 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase are reported on, from purification and isolation, to crystallogenesis, to experimental design and data collection and subsequent interpretation of results and novel insights gained.
ContributorsCoe, Jesse (Author) / Fromme, Petra (Thesis advisor) / Sayres, Scott (Thesis advisor) / Mujica, Vladimiro (Committee member) / Redding, Kevin (Committee member) / Arizona State University (Publisher)
Created2018
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
ABSTRACT
The catalytic chaperone of Rubisco is AAA+ protein Rubisco activase (Rca), which hydrolyzes ATP and thus undergoes conformational change, helping in reactivating Rubisco. Rca reactivates Rubisco plausibly by removing its C- terminal tail from the opening of its active site thus releasing the inhibitor, a sugar phosphate molecule. Rubisco and Rca are regulated by the stromal environment, which includes the ATP/ADP ratio, Mg2+ concentration, redox potential etc. Here the mechanistic regulation of tobacco β-Rca was studied using steady state enzyme kinetics in terms of product inhibition, Mg2+ activation, cooperativity and asymmetry. A continuous Pi measurement assay was developed, and using this assay catalytic parameters were obtained, such as kcat 20.6 ± 6.5 min-1 ( n = 9) and KM 0.113 ± 0.033 mM (n = 4). A Mg2+ induced increase of substrate affinity in Rca was observed, where the KM changes from 0.452 mM to 0.069 mM, with the changing of free Mg2+ concentration from 0.1 mM to 10 mM. Fitting the catalytic efficiency as a function free Mg2+ concentration by use of a binding model gave a Hill coefficient of 2.2, which indicates a secondary magnesium binding site on the enzyme. A 8.4 fold increase of catalytic efficiency with increasing magnesium from 0.1 mM to 6.5 mM suggests a significant Mg2+ induced regulation of Rca. Moderate product inhibition was observed in inhibition study (Ki = 0. 063 ± 0.018 mM). A positive cooperativity (nH = 2.1) in ATP hydrolysis between two subunits was observed in the presence of 0.132 mM ADP, but not in the absence of ADP. This indicated the presence of two different classes of subunits, suggesting an asymmetric model for the enzyme. Inhibited Rubisco (ER) up to 20 μM concentration did not affect ATPase activity, in line with previous reports. The concentration dependent correlation of Rca activity (tobacco β-Rca) and oligomerization (cotton β-Rca) suggested that the dimer maybe the most active oligomeric species. A nucleotide induced thermal stabilization of Rca was observed, where ADP is more stabilizing than ATP in the absence of Mg2+. Mg2+ has a small destabilizing effect alone and in presence of the ADP, but a stabilizing effect in presence of ATP. The ligand induced thermal stability was similar for cotton and tobacco β-Rca.
The catalytic chaperone of Rubisco is AAA+ protein Rubisco activase (Rca), which hydrolyzes ATP and thus undergoes conformational change, helping in reactivating Rubisco. Rca reactivates Rubisco plausibly by removing its C- terminal tail from the opening of its active site thus releasing the inhibitor, a sugar phosphate molecule. Rubisco and Rca are regulated by the stromal environment, which includes the ATP/ADP ratio, Mg2+ concentration, redox potential etc. Here the mechanistic regulation of tobacco β-Rca was studied using steady state enzyme kinetics in terms of product inhibition, Mg2+ activation, cooperativity and asymmetry. A continuous Pi measurement assay was developed, and using this assay catalytic parameters were obtained, such as kcat 20.6 ± 6.5 min-1 ( n = 9) and KM 0.113 ± 0.033 mM (n = 4). A Mg2+ induced increase of substrate affinity in Rca was observed, where the KM changes from 0.452 mM to 0.069 mM, with the changing of free Mg2+ concentration from 0.1 mM to 10 mM. Fitting the catalytic efficiency as a function free Mg2+ concentration by use of a binding model gave a Hill coefficient of 2.2, which indicates a secondary magnesium binding site on the enzyme. A 8.4 fold increase of catalytic efficiency with increasing magnesium from 0.1 mM to 6.5 mM suggests a significant Mg2+ induced regulation of Rca. Moderate product inhibition was observed in inhibition study (Ki = 0. 063 ± 0.018 mM). A positive cooperativity (nH = 2.1) in ATP hydrolysis between two subunits was observed in the presence of 0.132 mM ADP, but not in the absence of ADP. This indicated the presence of two different classes of subunits, suggesting an asymmetric model for the enzyme. Inhibited Rubisco (ER) up to 20 μM concentration did not affect ATPase activity, in line with previous reports. The concentration dependent correlation of Rca activity (tobacco β-Rca) and oligomerization (cotton β-Rca) suggested that the dimer maybe the most active oligomeric species. A nucleotide induced thermal stabilization of Rca was observed, where ADP is more stabilizing than ATP in the absence of Mg2+. Mg2+ has a small destabilizing effect alone and in presence of the ADP, but a stabilizing effect in presence of ATP. The ligand induced thermal stability was similar for cotton and tobacco β-Rca.
ContributorsHazra, Suratna (Author) / Wachter, Rebekka M. (Thesis advisor) / Fromme, Petra (Committee member) / Frasch, Wayne D (Committee member) / Arizona State University (Publisher)
Created2015
Description
Adenosine triphosphate (ATP) is the universal chemical energy currency in most living cells, used to power many cellular reactions and generated by an enzyme supercomplex known as the ATP synthase, consisting of a hydrophilic F1 subcomplex and a membrane-bound FO subcomplex. Driven by the electrochemical gradient generated by the respiratory or photosynthetic electron transport chain, the rotation of the FO domain drives movements of the central stalk in response to conformational changes in the F1 domain, in which the physical energy is converted into chemical energy through the condensation of ADP and Pi to ATP. The exact mechanism how ATP synthesis is coupled to proton translocation is not known as no structure of the intact ATP-synthase nor the intact FO subcomplex has been determined to date. Structural information may shed light on these mechanisms and aid in understanding how structural changed relate to its coupling to ATP synthesis. The work in this thesis has successful established a defined large-scale CF1FO isolation procedure resulting in high purity and high yield of this complex from spinach thylakoid membranes by incorporating a unique combination of biochemical methods will form the basis for the subsequent structural determination of this complex. Isolation began from the isolation of intact chloroplasts and the separation of intact thylakoid membranes. Both native and denaturing electrophoresis analyses clearly demonstrated that the purified CF1FO retains its quaternary structure consisting of the CF1 and CFO subcomplexes and nine subunits (five F1 subunits: α, β, γ, δ and ε, and four FO subunits: a, b, b' and c). Moreover, both ATP synthesis and hydrolysis activities were successfully detected using protein reconstitution in combination with acid-base incubation and in-gel ATPase assays, respectively. Furthermore, the ATP-synthase of H. modesticaldum, an anaerobic photosynthetic bacterium, was also isolated and characterized at the biochemical level. These biochemical characterizations directly influenced recent studies on the high-resolution structure determination of intact CF1FO using electron crystallography on two-dimensional crystals. The availability of the functionally intact CF1FO purified at a large scale will lead to studies that investigate the possible crystallization conditions to ultimately determine its three-dimensional structure at atomic resolution.
ContributorsYang, Jay-How (Author) / Fromme, Petra (Thesis advisor) / Redding, Kevin (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2015
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
The Heliobacterial reaction center (HbRC) is generally regarded as the most primitive photosynthetic reaction center (RC) known. Even if the HbRC is structurally and functionally simple compared to higher plants, the mechanisms of energy transduction preceding, inside the core, and from the RC are not totally established. Elucidating these structures and mechanisms are paramount to determining where the HbRC is in the grand scheme of RC evolution. In this work, the function and properties of the solubilized cyt c553, PetJ, were investigated, as well as the role HbRC localized menaquinone plays in light-induced electron transfer, and the interaction of the Nif-specific ferredoxin FdxB with reaction center particles devoid of bound FA/FB proteins. In chapter 2, I successfully express and purify a soluble version of PetJ that functions as a temperature dependent electron donor to P800+. Recombinant PetJ retains the spectroscopic characteristics of membrane-bound PetJ. The kinetics were characteristic of a bimolecular reaction with a second order rate of 1.53 x 104 M-1s-1 at room temperature and a calculated activation energy of 91 kJ/mol. In chapter 4, I use reverse phase high-performance liquid chromatography (HPLC) to detect the light-induced generation of Menaquinol-9 (MQH2) in isolated heliobacterial membranes. This process is dependent on laser power, pH, temperature, and can be modified by the presence of the artificial electron acceptor benzyl viologen (BV) and the inhibitors azoxystrobin and terbutryn. The addition of the bc complex inhibitor azoxystrobin decreases the ratio of MQ to MQH2. This indicates competition between the HbRC and the bc complex, and hints toward a truncated cyclic electron flow pathway. In chapter 5, the Nif-Specific ferredoxin FdxB was recombinantly expressed and shown to oxidize the terminal cofactor in the HbRC, FX-, in a concentration-dependent manner. This work indicates the HbRC may be able to reduce a wide variety of electron acceptors that may be involved in specific metabolic processes.
ContributorsKashey, Trevor (Author) / Redding, Kevin E (Thesis advisor) / Fromme, Petra (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2015
Description
Viral protein U (Vpu) is a type-III integral membrane protein encoded by the Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays vital roles in down-regulation of CD4 receptors in T cells and also in the budding of virions. But, there remain key structure/function questions regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis and thus, it makes for an attractive target to study the structural attributes of this protein by elucidating a structural model with X-ray crystallography. This study describes a multi-pronged approach of heterologous over-expression of Vpu. The strategies of purification and biophysical/ biochemical characterization of the different versions of the protein to evaluate their potential for crystallization are also detailed. Furthermore, various strategies employed for the crystallization of Vpu by both in surfo and in cubo techniques, and the challenges faced towards the structural studies of this membrane protein by characterization with solution Nuclear magnetic resonance (NMR) spectroscopy are also described.
ContributorsDeb, Arpan (Author) / Leket-Mor, Tsafrir S (Thesis advisor) / Fromme, Petra (Committee member) / Mason, Hugh (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2016