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- All Subjects: Biochemistry
- Genre: Doctoral Dissertation
- Creators: Fromme, Petra
- Creators: Levitus, Marcia
Description
There is a critical need for the development of clean and efficient energy sources. Hydrogen is being explored as a viable alternative to fuels in current use, many of which have limited availability and detrimental byproducts. Biological photo-production of H2 could provide a potential energy source directly manufactured from water and sunlight. As a part of the photosynthetic electron transport chain (PETC) of the green algae Chlamydomonas reinhardtii, water is split via Photosystem II (PSII) and the electrons flow through a series of electron transfer cofactors in cytochrome b6f, plastocyanin and Photosystem I (PSI). The terminal electron acceptor of PSI is ferredoxin, from which electrons may be used to reduce NADP+ for metabolic purposes. Concomitant production of a H+ gradient allows production of energy for the cell. Under certain conditions and using the endogenous hydrogenase, excess protons and electrons from ferredoxin may be converted to molecular hydrogen. In this work it is demonstrated both that certain mutations near the quinone electron transfer cofactor in PSI can speed up electron transfer through the PETC, and also that a native [FeFe]-hydrogenase can be expressed in the C. reinhardtii chloroplast. Taken together, these research findings form the foundation for the design of a PSI-hydrogenase fusion for the direct and continuous photo-production of hydrogen in vivo.
ContributorsReifschneider, Kiera (Author) / Redding, Kevin (Thesis advisor) / Fromme, Petra (Committee member) / Jones, Anne (Committee member) / Arizona State University (Publisher)
Created2013
Description
Solution conformations and dynamics of proteins and protein-DNA complexes are often difficult to predict from their crystal structures. The crystal structure only shows a snapshot of the different conformations these biological molecules can have in solution. Multiple different conformations can exist in solution and potentially have more importance in the biological activity. DNA sliding clamps are a family of proteins with known crystal structures. These clamps encircle the DNA and enable other proteins to interact more efficiently with the DNA. Eukaryotic PCNA and prokaryotic β clamp are two of these clamps, some of the most stable homo-oligomers known. However, their solution stability and conformational equilibrium have not been investigated in depth before. Presented here are the studies involving two sliding clamps: yeast PCNA and bacterial β clamp. These studies show that the β clamp has a very different solution stability than PCNA. These conclusions were reached through various different fluorescence-based experiments, including fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), single molecule fluorescence, and various time resolved fluorescence techniques. Interpretations of these, and all other, fluorescence-based experiments are often affected by the properties of the fluorophores employed. Often the fluorescence properties of these fluorophores are influenced by their microenvironments. Fluorophores are known to sometimes interact with biological molecules, and this can have pronounced effects on the rotational mobility and photophysical properties of the dye. Misunderstanding the effect of these photophysical and rotational properties can lead to a misinterpretation of the obtained data. In this thesis, photophysical behaviors of various organic dyes were studied in the presence of deoxymononucleotides to examine more closely how interactions between fluorophores and DNA bases can affect fluorescent properties. Furthermore, the properties of cyanine dyes when bound to DNA and the effect of restricted rotation on FRET are presented in this thesis. This thesis involves studying fluorophore photophysics in various microenvironments and then expanding into the solution stability and dynamics of the DNA sliding clamps.
ContributorsRanjit, Suman (Author) / Levitus, Marcia (Thesis advisor) / Lindsay, Stuart (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2013
Description
The need for a renewable and sustainable light-driven energy source is the motivation for this work, which utilizes a challenging, yet practical and attainable bio-inspired approach to develop an artificial oxygen evolving complex, which builds upon the principles of the natural water splitting mechanism in oxygenic photosynthesis. In this work, a stable framework consisting of a three-dimensional DNA tetrahedron has been used for the design of a bio-mimic of the Oxygen-Evolving Complex (OEC) found in natural Photosystem II (PSII). PSII is a large protein complex that evolves all the oxygen in the atmosphere, but it cannot be used directly in artificial systems, as the light reactions lead to damage of one of Photosystem II's core proteins, D1, which has to be replaced every half hour in the presence of sunlight. The final goal of the project aims to build the catalytic center of the OEC, including the Mn4CaCl metal cluster and its protein environment in the stable DNA framework of a tetrahedron, which can subsequently be connected to a photo-stable artificial reaction center that performs light-induced charge separation. Regions of the peptide sequences containing Mn4CaCl ligation sites are implemented in the design of the aOEC (artificial oxygen-evolving complex) and are attached to sites within the tetrahedron to facilitate assembly. Crystals of the tetrahedron have been obtained, and X-ray crystallography has been used for characterization. As a proof of concept, metal-binding peptides have been coupled to the DNA tetrahedron which allowed metal-containing porphyrins, specifically Fe(III) meso-Tetra(4-sulfonatophenyl) porphyrin chloride, to be encapsulated inside the DNA-tetrahedron. The porphyrins were successfully assembled inside the tetrahedron through coordination of two terminal histidines from the orthogonally oriented peptides covalently attached to the DNA. The assembly has been characterized using Electron Paramagnetic Resonance (EPR), optical spectroscopy, Dynamic Light Scattering (DLS), and x-ray crystallography. The results reveal that the spin state of the metal, iron (III), switches during assembly from the high-spin state to low-spin state.
ContributorsRendek, Kimberly Nicole (Author) / Fromme, Petra (Thesis advisor) / Chen, Julian (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2012
Description
Spider dragline silk is well known for its outstanding mechanical properties - a combination of strength and extensibility that makes it one of the toughest materials known. Two proteins, major ampullate spidroin 1 (MaSp1) and 2 (MaSp2), comprise dragline silk fibers. There has been considerable focus placed on understanding the source of spider silk's unique mechanical properties by investigating the protein composition, molecular structure and dynamics. Chemical compositional heterogeneity of spider silk fiber is critical to understand as it provides important information for the interactions between MaSp1 and MaSp2. Here, the amino acid composition of dragline silk protein was precisely determined using a solution-state nuclear magnetic resonance (NMR) approach on hydrolyzed silk fibers. In a similar fashion, solution-state NMR was applied to probe the "13"C/"15"N incorporation in silk, which is essential to understand for designing particular solid-state NMR methods for silk structural characterization. Solid-state NMR was used to elucidate silk protein molecular dynamics and the supercontraction mechanism. A "2"H-"13"C heteronuclear correlation (HETCOR) solid-state NMR technique was developed to extract site-specific "2"H quadrupole patterns and spin-lattice relaxation rates for understanding backbone and side-chain dynamics. Using this technique, molecular dynamics were determined for a number of repetitive motifs in silk proteins - Ala residing nanocrystalline &beta-sheet; domains, 3"1"-helical regions, and, Gly-Pro-Gly-XX &beta-turn; motifs. The protein backbone and side-chain dynamics of silk fibers in both dry and wet states reveal the impact of water on motifs with different secondary structures. Spider venom is comprised of a diverse range of molecules including salts, small organics, acylpolyamines, peptides and proteins. Neurotoxins are an important family of peptides in spider venom and have been shown to target and modulate various ion channels. The neurotoxins are Cys-rich and share an inhibitor Cys knot (ICK) fold. Here, the molecular structure of one G. rosea tarantula neurotoxin, GsAF2, was determined by solution-state NMR. In addition, the interaction between neurotoxins and model lipid bilayers was probed with solid-state NMR and negative-staining (NS) transmission electron microscopy (TEM). It is shown that the neurotoxins influence lipid bilayer assembly and morphology with the formation of nanodiscs, worm-like micelles and small vesicles.
ContributorsShi, Xiangyan (Author) / Yarger, Jeffery L (Thesis advisor) / Holland, Gregory P (Thesis advisor) / Levitus, Marcia (Committee member) / Marzke, Robert F (Committee member) / Arizona State University (Publisher)
Created2014
Description
Rapid and reliable separation and analysis of proteins require powerful analytical methods. The analysis of proteins becomes especially challenging when only small sample volumes are available, concomitantly with low concentrations of proteins. Time critical situations pose additional challenges. Due to these challenges, conventional macro-scale separation techniques reach their limitations. While microfluidic devices require only pL-nL sample volumes, they offer several advantages such as speed, efficiency, and high throughput. This work elucidates the capability to manipulate proteins in a rapid and reliable manner with a novel migration technique, namely dielectrophoresis (DEP). Since protein analysis can often be achieved through a combination of orthogonal techniques, adding DEP as a gradient technique to the portfolio of protein manipulation methods can extend and improve combinatorial approaches. To this aim, microfluidic devices tailored with integrated insulating obstacles were fabricated to create inhomogeneous electric fields evoking insulator-based DEP (iDEP). A main focus of this work was the development of pre-concentration devices where topological micropost arrays are fabricated using standard photo- and soft lithographic techniques. With these devices, positive DEP-driven streaming of proteins was demonstrated for the first time using immunoglobulin G (IgG) and bovine serum albumin. Experimentally observed iDEP concentrations of both proteins were in excellent agreement with positive DEP concentration profiles obtained by numerical simulations. Moreover, the micropost iDEP devices were improved by introducing nano-constrictions with focused ion beam milling with which numerical simulations suggested enhancement of the DEP effect, leading to a 12-fold increase in concentration of IgG. Additionally, concentration of β-galactosidase was observed, which seems to occur due to an interplay of negative DEP, electroosmosis, electrokinesis, diffusion, and ion concentration polarization. A detailed study was performed to investigate factors influencing protein DEP under DC conditions, including electroosmosis, electrophoresis, and Joule heating. Specifically, temperature rise within the iDEP device due to Joule heating was measured experimentally with spatial and temporal resolution by employing the thermosensitive dye Rhodamine B. Unlike DNA and cells, protein DEP behavior is not well understood to date. Therefore, this detailed study of protein DEP provides novel information to eventually optimize this protein migration method for pre-concentration, separation, and fractionation.
ContributorsNakano, Asuka (Author) / Ros, Alexandra (Thesis advisor) / Hayes, Mark (Committee member) / Levitus, Marcia (Committee member) / Arizona State University (Publisher)
Created2014
Description
Photosynthesis is the primary source of energy for most living organisms. Light harvesting complexes (LHC) play a vital role in harvesting sunlight and passing it on to the protein complexes of the electron transfer chain which create the electrochemical potential across the membrane which drives ATP synthesis. phycobilisomes (PBS) are the most important LHCs in cyanobacteria. PBS is a complex of three light harvesting proteins: phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (APC). This work has been done on a newly discovered cyanobacterium called Leptolyngbya Heron Island (L.HI). This study has three important goals: 1) Sequencing, assembly and annotation of the L.HI genome - Since this is a newly discovered cyanobacterium, its genome was not previously elucidated. Illumina sequencing, a type of next generation sequencing (NGS) technology was employed to sequence the genome. Unfortunately, the natural isolate contained other contaminating and potentially symbiotic bacterial populations. A novel bioinformatics strategy for separating DNA from contaminating bacterial populations from that of L.HI was devised which involves a combination of tetranucleotide frequency, %(G+C), BLAST analysis and gene annotation. 2) Structural elucidation of phycoerythrin - Phycoerythrin is the most important protein in the PBS assembly because it is one of the few light harvesting proteins which absorbs green light. The protein was crystallized and its structure solved to a resolution of 2Å. This protein contains two chemically distinct types of chromophores: phycourobilin and phycoerythrobilin. Energy transfer calculations indicate that there is unidirectional flow of energy from phycourobilin to phycoerythrobilin. Energy transfer time constants using Forster energy transfer theory have been found to be consistent with experimental data available in literature. 3) Effect of chromatic acclimation on photosystems - Chromatic acclimation is a phenomenon in which an organism modulates the ratio of PE/PC with change in light conditions. Our investigation in case of L.HI has revealed that the PE is expressed more in green light than PC in red light. This leads to unequal harvesting of light in these two states. Therefore, photosystem II expression is increased in red-light acclimatized cells coupled with an increase in number of PBS.
ContributorsPaul, Robin (Author) / Fromme, Petra (Thesis advisor) / Ros, Alexandra (Committee member) / Roberson, Robert (Committee member) / Arizona State University (Publisher)
Created2014
Description
Membrane proteins are a vital part of cellular structure. They are directly involved in many important cellular functions, such as uptake, signaling, respiration, and photosynthesis, among others. Despite their importance, however, less than 500 unique membrane protein structures have been determined to date. This is due to several difficulties with macromolecular crystallography, primarily the difficulty of growing large, well-ordered protein crystals. Since the first proof of concept for femtosecond nanocrystallography showing that diffraction patterns can be collected on extremely small crystals, thus negating the need to grow larger crystals, there have been many exciting advancements in the field. The technique has been proven to show high spatial resolution, thus making it a viable method for structural biology. However, due to the ultrafast nature of the technique, which allows for a lack of radiation damage in imaging, even more interesting experiments are possible, and the first temporal and spatial images of an undamaged structure could be acquired. This concept was denoted as time-resolved femtosecond nanocrystallography.
This dissertation presents on the first time-resolved data set of Photosystem II where structural changes can actually be seen without radiation damage. In order to accomplish this, new crystallization techniques had to be developed so that enough crystals could be made for the liquid jet to deliver a fully hydrated stream of crystals to the high-powered X-ray source. These changes are still in the preliminary stages due to the slightly lower resolution data obtained, but they are still a promising show of the power of this new technique. With further optimization of crystal growth methods and quality, injection technique, and continued development of data analysis software, it is only a matter of time before the ability to make movies of molecules in motion from X-ray diffraction snapshots in time exists. The work presented here is the first step in that process.
This dissertation presents on the first time-resolved data set of Photosystem II where structural changes can actually be seen without radiation damage. In order to accomplish this, new crystallization techniques had to be developed so that enough crystals could be made for the liquid jet to deliver a fully hydrated stream of crystals to the high-powered X-ray source. These changes are still in the preliminary stages due to the slightly lower resolution data obtained, but they are still a promising show of the power of this new technique. With further optimization of crystal growth methods and quality, injection technique, and continued development of data analysis software, it is only a matter of time before the ability to make movies of molecules in motion from X-ray diffraction snapshots in time exists. The work presented here is the first step in that process.
ContributorsKupitz, Christopher (Author) / Fromme, Petra (Thesis advisor) / Spence, John C. (Thesis advisor) / Redding, Kevin (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2014
Description
Ribulose-1, 5-bisphosphate carboxylase oxygenase, commonly known as RuBisCO, is an enzyme involved in carbon fixation in photosynthetic organisms. The enzyme is subject to a mechanism-based deactivation during its catalytic cycle. RuBisCO activase (Rca), an ancillary enzyme belonging to the AAA+ family of the ATP-ases, rescues RuBisCO by facilitating the removal of the tightly bound sugar phosphates from the active sites of RuBisCO. In this work, we investigated the ATP/ADP dependent oligomerization equilibrium of fluorescently tagged Rca for a wide range of concentrations using fluorescence correlation spectroscopy. Results show that in the presence of ADP-Mg2+, the oligomerization state of Rca gradually changes in steps of two subunits. The most probable association model supports the dissociation constants (K_d) of ∼4, 1, 1 μM for the monomer-dimer, dimer-tetramer, and tetramer-hexamer equlibria, respectively. Rca continues to assemble at higher concentrations which are indicative of the formation of aggregates. In the presence of ATP-Mg2+, a similar stepwise assembly is observed. However, at higher concentrations (30-75 µM), the average oligomeric size remains relatively unchanged around six subunits per oligomer. This is in sharp contrast with observations in ADP-Mg2+, where a marked decrease in the diffusion coefficient of Rca was observed, consistent with the formation of aggregates. The estimated K_d values obtained from the analysis of the FCS decays were similar for the first steps of the assembly process in both ADP-Mg2+ and ATP-Mg2+. However, the formation of the hexamer from the tetramer is much more favored in ATP-Mg2+, as evidenced from 20 fold lower K_d associated with this assembly step. This suggests that the formation of a hexameric ring in the presence of ATP-Mg2+. In addition to that, Rca aggregation is largely suppressed in the presence of ATP-Mg2+, as evidenced from the 1000 fold larger K_d value for the hexamer-24 mer association step. In essence, a fluorescence-based method was developed to monitor in vitro protein oligomerization and was successfully applied with Rca. The results provide a strong hint at the active oligomeric structure of Rca, and this information will hopefully help the ongoing research on the mechanistic enzymology of Rca.
ContributorsChakraborty, Manas (Author) / Levitus, Marcia (Thesis advisor) / Angell, Charles (Committee member) / Lindsay, Stuart (Committee member) / Arizona State University (Publisher)
Created2014
Description
Proteins and peptides fold into dynamic structures that access a broad functional landscape, however, designing artificial polypeptide systems continues to be a great chal-lenge. Conversely, deoxyribonucleic acid (DNA) engineering is now routinely used to build a wide variety of two dimensional and three dimensional (3D) nanostructures from simple hybridization based rules, and their functional diversity can be significantly ex-panded through site specific incorporation of the appropriate guest molecules. This dis-sertation describes a gentle methodology for using short (8 nucleotide) peptide nucleic acid (PNA) linkers to assemble polypeptides within a 3D DNA nanocage, as a proof of concept for constructing artificial catalytic centers. PNA-polypeptide conjugates were synthesized directly using microwave assisted solid phase synthesis or alternatively PNA linkers were conjugated to biologically expressed proteins using chemical crosslinking. The PNA-polypeptides hybridized to the preassembled DNA nanocage at room tempera-ture or 11 ⁰C and could be assembled in a stepwise fashion. Time resolved fluorescence anisotropy and gel electrophoresis were used to determine that a negatively charged az-urin protein was repelled outside of the negatively charged DNA nanocage, while a posi-tively charged cytochrome c protein was retained inside. Spectroelectrochemistry and an in-gel luminol oxidation assay demonstrated the cytochrome c protein remained active within the DNA nanocage and its redox potential decreased modestly by 10 mV due to the presence of the DNA nanocage. These results demonstrate the benign PNA assembly conditions are ideal for preserving polypeptide structure and function, and will facilitate the polypeptide-based assembly of artificial catalytic centers inside a stable DNA nanocage. A prospective application of assembling multiple cyclic γ-PNA-peptides to mimic the oxygen-evolving complex (OEC) catalytic active site from photosystem II (PSII) is described. In this way, the robust catalytic capacity of PSII could be utilized, without suffering the light-induced damage that occurs by the photoreactions within PSII via triplet state formation, which limits the efficiency of natural photosynthesis. There-fore, this strategy has the potential to revolutionize the process of designing and building robust catalysts by leveraging nature's recipes, and also providing a flexible and con-trolled artificial environment that might even improve them further towards commercial viability.
ContributorsFlory, Justin David (Author) / Fromme, Petra (Thesis advisor) / Yan, Hao (Committee member) / Buttry, Daniel (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2014
Description
Biophysical techniques have been increasingly applied toward answering biological questions with more precision. Here, three different biological systems were studied with the goal of understanding their dynamic differences, either conformational dynamics within the system or oligomerization dynamics between monomers. With Cy3 on the 5' end of DNA, the effects of changing the terminal base pair were explored using temperature-dependent quantum yields. It was discovered, in combination with simulations, that a terminal thymine base has the weakest stacking interactions with the Cy3 dye compared to the other three bases. With ME1 heterodimers, the goal was to see if engineering a salt bridge at the dimerization interface could allow for control over dimerization in a pH-dependent manner. This was performed experimentally by measuring FRET between monomers containing either a Dap or an Asp mutation and comparing FRET efficiency at different pHs. It was demonstrated that the heterodimeric salt bridge would only form in a pH range near neutrality. Finally, with DNA processivity clamps, one aim was to compare the equilibrium dissociation constants, kinetic rate constants, and lifetimes of the closed rings for beta clamp and PCNA. This was done using a variety of biophysical techniques but with three as the main focus: fluorescence correlation spectroscopy, single-molecule experiments, and time-correlated single photon counting measurements. The stability of beta clamp was found to be three orders of magnitude higher when measuring solution stability but only one order of magnitude higher when measuring intrinsic stability, which is a result of salt bridge interactions in the interface of beta clamp. Ongoing work built upon the findings from this project by attempting to disrupt interface stability of different beta clamp mutants by adding salt or changing the pH of the solution. Lingering questions about the dynamics of different areas of the clamps has led to another project for which we have developed a control to demystify some unexpected similarities between beta clamp mutants. With that project, we show that single-labeled and double-labeled samples have similar autocorrelation decays in florescence correlation spectroscopy, allowing us to rule out the dyes themselves as causing fluctuations in the 10-100 microsecond timescale.
ContributorsBinder, Jennifer (Author) / Levitus, Marcia (Thesis advisor) / Wachter, Rebekka (Committee member) / Ros, Robert (Committee member) / Arizona State University (Publisher)
Created2015