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Description
The biological and chemical diversity of protein structure and function can be greatly expanded by position-specific incorporation of non-natural amino acids bearing a variety of functional groups. Non-cognate amino acids can be incorporated into proteins at specific sites by using orthogonal aminoacyl-tRNA synthetase/tRNA pairs in conjunction with nonsense, rare, or 4-bp codons. There has been considerable progress in developing new types of amino acids, in identifying novel methods of tRNA aminoacylation, and in expanding the genetic code to direct their position. Chemical aminoacylation of tRNAs is accomplished by acylation and ligation of a dinucleotide (pdCpA) to the 3'-terminus of truncated tRNA. This strategy allows the incorporation of a wide range of natural and unnatural amino acids into pre-determined sites, thereby facilitating the study of structure-function relationships in proteins and allowing the investigation of their biological, biochemical and biophysical properties. Described in Chapter 1 is the current methodology for synthesizing aminoacylated suppressor tRNAs. Aminoacylated suppressor tRNACUAs are typically prepared by linking pre-aminoacylated dinucleotides (aminoacyl-pdCpAs) to 74 nucleotide (nt) truncated tRNAs (tRNA-COH) via a T4 RNA ligase mediated reaction. Alternatively, there is another route outlined in Chapter 1 that utilizes a different pre-aminoacylated dinucleotide, AppA. This dinucleotide has been shown to be a suitable substrate for T4 RNA ligase mediated coupling with abbreviated tRNA-COHs for production of 76 nt aminoacyl-tRNACUAs. The synthesized suppressor tRNAs have been shown to participate in protein synthesis in vitro, in an S30 (E. coli) coupled transcription-translation system in which there is a UAG codon in the mRNA at the position corresponding to Val10. Chapter 2 describes the synthesis of two non-proteinogenic amino acids, L-thiothreonine and L-allo-thiothreonine, and their incorporation into predetermined positions of a catalytically competent dihydrofolate reductase (DHFR) analogue lacking cysteine. Here, the elaborated proteins were site-specifically derivitized with a fluorophore at the thiothreonine residue. The synthesis and incorporation of phosphorotyrosine derivatives into DHFR is illustrated in Chapter 3. Three different phosphorylated tyrosine derivatives were prepared: bis-nitrobenzylphosphoro-L-tyrosine, nitrobenzylphosphoro-L-tyrosine, and phosphoro-L-tyrosine. Their ability to participate in a protein synthesis system was also evaluated.
ContributorsNangreave, Ryan Christopher (Author) / Hecht, Sidney M. (Thesis advisor) / Yan, Hao (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2013
Description
Alzheimer's Disease (AD) is the sixth leading cause of death in the United States and the most common form of dementia. Its cause remains unknown, but it is known to involve two hallmark pathologies: Amyloid Beta plaques and neurofibrillary tangles (NFTs). Several proteins have been implicated in the formation of neurofibrillary tangles, including Tau and S100B. S100B is a dimeric protein that is typically found bound to Ca(II) or Zn(II). These experiments relate to the involvement of S100B in Alzheimer's Disease-related processes and the results suggest that future research of S100B is warranted. Zn(II)-S100B was found to increase the rate at which tau assembled into paired helical filaments, as well as affect the rate at which tubulin polymerized into microtubules and the morphology of SH-SY5Y neuroblastoma cells after 72 hours of incubation. Zn(II)-S100B also increased the firing rate of hippocampal neurons after 36 hours of incubation. Together, these results suggest several possibilities: Zn(II)-S100B may be a key part of the formation of paired helical filaments (PHFs) that subsequently form NFTs. Zn(II)-S100B may also be competing with tau to bind tubulin, which could lead to an instability of microtubules and subsequent cell death. This finding aligns with the neurodegeneration that is commonly seen in AD and which could be a result of this microtubule instability. Ultimately, these results suggest that S100B is likely involved in several AD-related processes, and if the goal is to find an efficient and effective therapeutic target for AD, the relationship between S100B, particularly Zn(II)-S100B, and tau needs to be further studied.
ContributorsNaegele, Hayley (Author) / Mcgregor, Wade C (Thesis advisor) / Baluch, Debra (Committee member) / Francisco, Wilson (Committee member) / Arizona State University (Publisher)
Created2013
Description
ABSTRACT
Post Translational Modifications (PTMs) are a series of chemical modifications with the capacity to expand the structural and functional repertoire of proteins. PTMs can regulate protein-protein interaction, localization, protein turn-over, the active state of the protein, and much more. This can dramatically affect cell processes as relevant as gene expression, cell-cell recognition, and cell signaling. Along these lines, this Ph.D. thesis examines the role of two of the most important PTMs: glycosylation and phosphorylation.
In chapters 2, 3 and 4, a 10,000 peptide microarray is used to analyze the glycan variations in a series lipopolysaccharides (LPS) from Gram negative bacteria. This research was the first to demonstrate that using a small subset of random sequence peptides, it was possible to identify a small subset with the capacity to bind to the LPS of bacteria. These peptides bound to LPS not only in the solid surface of the array but also in solution as demonstrated with surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and flow cytometry. Interestingly, some of the LPS binding peptides also exhibit antimicrobial activity, a property that is also analyzed in this work.
In chapters 5 and 6, the role of protein phosphorylation, another PTM, is analyzed in the context of human cancer. High risk neuroblastoma, a very aggressive pediatric cancer, was studied with emphasis on the phosphorylations of two selected oncoproteins: the transcription factor NMYC and the adaptor protein ShcC. Both proteins were isolated from high risk neuroblastoma cells, and a targeted-directed tandem mass spectrometry (LC-MS/MS) methodology was used to identify the phosphorylation sites in each protein. Using this method dramatically improved the phosphorylation site detection and increased the number of sites detected up to 250% in comparison with previous studies. Several of the novel identified sites were located in functional domain of the proteins and that some of them are homologous to known active sites in other proteins of the same family. The chapter concludes with a computational prediction of the kinases that potentially phosphorylate those sites and a series of assays to show this phosphorylation occurred in vitro.
Post Translational Modifications (PTMs) are a series of chemical modifications with the capacity to expand the structural and functional repertoire of proteins. PTMs can regulate protein-protein interaction, localization, protein turn-over, the active state of the protein, and much more. This can dramatically affect cell processes as relevant as gene expression, cell-cell recognition, and cell signaling. Along these lines, this Ph.D. thesis examines the role of two of the most important PTMs: glycosylation and phosphorylation.
In chapters 2, 3 and 4, a 10,000 peptide microarray is used to analyze the glycan variations in a series lipopolysaccharides (LPS) from Gram negative bacteria. This research was the first to demonstrate that using a small subset of random sequence peptides, it was possible to identify a small subset with the capacity to bind to the LPS of bacteria. These peptides bound to LPS not only in the solid surface of the array but also in solution as demonstrated with surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and flow cytometry. Interestingly, some of the LPS binding peptides also exhibit antimicrobial activity, a property that is also analyzed in this work.
In chapters 5 and 6, the role of protein phosphorylation, another PTM, is analyzed in the context of human cancer. High risk neuroblastoma, a very aggressive pediatric cancer, was studied with emphasis on the phosphorylations of two selected oncoproteins: the transcription factor NMYC and the adaptor protein ShcC. Both proteins were isolated from high risk neuroblastoma cells, and a targeted-directed tandem mass spectrometry (LC-MS/MS) methodology was used to identify the phosphorylation sites in each protein. Using this method dramatically improved the phosphorylation site detection and increased the number of sites detected up to 250% in comparison with previous studies. Several of the novel identified sites were located in functional domain of the proteins and that some of them are homologous to known active sites in other proteins of the same family. The chapter concludes with a computational prediction of the kinases that potentially phosphorylate those sites and a series of assays to show this phosphorylation occurred in vitro.
ContributorsMorales Betanzos, Carlos (Author) / LaBaer, Joshua (Thesis advisor) / Allen, James (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2014
Description
Protein-surface interactions, no matter structured or unstructured, are important in both biological and man-made systems. Unstructured interactions are more difficult to study with conventional techniques due to the lack of a specific binding structure. In this dissertation, a novel approach is employed to study the unstructured interactions between proteins and heterogonous surfaces, by looking at a large number of different binding partners at surfaces and using the binding information to understand the chemistry of binding. In this regard, surface-bound peptide arrays are used as a model for the study. Specifically, in Chapter 2, the effects of charge, hydrophobicity and length of surface-bound peptides on binding affinity for specific globular proteins (&beta-galactosidase and &alpha1-antitrypsin) and relative binding of different proteins were examined with LC Sciences peptide array platform. While the general charge and hydrophobicity of the peptides are certainly important, more surprising is that &beta-galactosidase affinity for the surface does not simply increase with the length of the peptide. Another interesting observation that leads to the next part of the study is that even very short surface-bound peptides can have both strong and selective interactions with proteins. Hence, in Chapter 3, selected tetrapeptide sequences with known binding characteristics to &beta-galactosidase are used as building blocks to create longer sequences to see if the binding function can be added together. The conclusion is that while adding two component sequences together can either greatly increase or decrease overall binding and specificity, the contribution to the binding affinity and specificity of the individual binding components is strongly dependent on their position in the peptide. Finally, in Chapter 4, another array platform is utilized to overcome the limitations associated with LC Sciences. It is found that effects of peptide sequence properties on IgG binding with HealthTell array are quiet similar to what was observed with &beta-galactosidase on LC Science array surface. In summary, the approach presented in this dissertation can provide binding information for both structured and unstructured interactions taking place at complex surfaces and has the potential to help develop surfaces covered with specific short peptide sequences with relatively specific protein interaction profiles.
ContributorsWang, Wei (Author) / Woodbury, Neal W (Thesis advisor) / Liu, Yan (Committee member) / Chaput, John (Committee member) / Arizona State University (Publisher)
Created2014
Description
Natural hydrogenases catalyze the reduction of protons to molecular hydrogen reversibly under mild conditions; these enzymes have an unusual active site architecture, in which a diiron site is connected to a cubane type [4Fe-4S] cluster. Due to the relevance of this reaction to energy production, and in particular to sustainable fuel production, there have been substantial amount of research focused on developing biomimetic organometallic models. However, most of these organometallic complexes cannot revisit the structural and functional fine-tuning provided by the protein matrix as seen in the natural enzyme. The goal of this thesis is to build a protein based functional mimic of [Fe-Fe] hydrogenases. I used a 'retrosynthetic' approach that separates out two functional aspects of the natural enzyme. First, I built an artificial electron transfer domain by engineering two [4Fe-4S] cluster binding sites into an existing protein, DSD, which is a de novo designed domain swapped dimer. The resulting protein, DSD-bis[4Fe-4S], contains two clusters at a distance of 36 Å . I then varied distance between two clusters using vertical translation along the axis of the coiled coil; the resulting protein demonstrates efficient electron transfer to/from redox sites. Second, I built simple, functional artificial hydrogenases by using an artificial amino acid comprising a 1,3 dithiol moiety to anchor a biomimetic [Fe-Fe] active site within the protein scaffold Correct incorporation of the cluster into a model helical peptide was verified by UV-Vis, FTIR, ESI-MS and CD spectroscopy. This synthetic strategy is extended to the de novo design of more complex protein architectures, four-helix bundles that host the di-iron cluster within the hydrophobic core. In a separate approach, I developed a generalizable strategy to introduce organometallic catalytic sites into a protein scaffold. I introduced a biomimetic organometallic complex for proton reduction by covalent conjugation to biotin. The streptavidin-bound complex is significantly more efficient in photocatalytic hydrogen production than the catalyst alone. With these artificial proteins, it will be possible to explore the effect of second sphere interactions on the activity of the diiron center, and to include in the design properties such as compatibility with conductive materials and electrodes.
ContributorsRoy, Anindya (Author) / Ghirlanda, Giovanna (Thesis advisor) / Yan, Hao (Committee member) / Gust, Devens (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
Redox reactions are crucial to energy transduction in biology. Protein film electrochemistry (PFE) is a technique for studying redox proteins in which the protein is immobilized at an electrode surface so as to allow direct exchange of electrons. Establishing a direct electronic connection eliminates the need for redoxactive mediators, thus allowing for interrogation of the redox protein of interest. PFE has proven a versatile tool that has been used to elucidate the properties of many technologically relevant redox proteins including hydrogenases, laccases, and glucose oxidase.
This dissertation is comprised of two parts: extension of PFE to a novel electrode material and application of PFE to the investigation of a new type of hydrogenase. In the first part, mesoporous antimony-doped tin oxide (ATO) is employed for the first time as an electrode material for protein film electrochemistry. Taking advantage of the excellent optical transparency of ATO, spectroelectrochemistry of cytochrome c is demonstrated. The electrochemical and spectroscopic properties of the protein are analogous to those measured for the native protein in solution, and the immobilized protein is stable for weeks at high loadings. In the second part, PFE is used to characterize the catalytic properties of the soluble hydrogenase I from Pyrococcus furiosus (PfSHI). Since this protein is highly thermostable, the temperature dependence of catalytic properties was investigated. I show that the preference of the enzyme for reduction of protons (as opposed to oxidation of hydrogen) and the reactions with oxygen are highly dependent on temperature, and the enzyme is tolerant to oxygen during both oxidative and reductive catalysis.
This dissertation is comprised of two parts: extension of PFE to a novel electrode material and application of PFE to the investigation of a new type of hydrogenase. In the first part, mesoporous antimony-doped tin oxide (ATO) is employed for the first time as an electrode material for protein film electrochemistry. Taking advantage of the excellent optical transparency of ATO, spectroelectrochemistry of cytochrome c is demonstrated. The electrochemical and spectroscopic properties of the protein are analogous to those measured for the native protein in solution, and the immobilized protein is stable for weeks at high loadings. In the second part, PFE is used to characterize the catalytic properties of the soluble hydrogenase I from Pyrococcus furiosus (PfSHI). Since this protein is highly thermostable, the temperature dependence of catalytic properties was investigated. I show that the preference of the enzyme for reduction of protons (as opposed to oxidation of hydrogen) and the reactions with oxygen are highly dependent on temperature, and the enzyme is tolerant to oxygen during both oxidative and reductive catalysis.
ContributorsKwan, Patrick Karchung (Author) / Jones, Anne K (Thesis advisor) / Francisco, Wilson (Committee member) / Moore, Thomas (Committee member) / Arizona State University (Publisher)
Created2014
Description
ABSTRACT
X-Ray crystallography and NMR are two major ways of achieving atomic
resolution of structure determination for macro biomolecules such as proteins. Recently, new developments of hard X-ray pulsed free electron laser XFEL opened up new possibilities to break the dilemma of radiation dose and spatial resolution in diffraction imaging by outrunning radiation damage with ultra high brightness femtosecond X-ray pulses, which is so short in time that the pulse terminates before atomic motion starts. A variety of experimental techniques for structure determination of macro biomolecules is now available including imaging of protein nanocrystals, single particles such as viruses, pump-probe experiments for time-resolved nanocrystallography, and snapshot wide- angle x-ray scattering (WAXS) from molecules in solution. However, due to the nature of the "diffract-then-destroy" process, each protein crystal would be destroyed once
probed. Hence a new sample delivery system is required to replenish the target crystal at a high rate. In this dissertation, the sample delivery systems for the application of XFELs to biomolecular imaging will be discussed and the severe challenges related to the delivering of macroscopic protein crystal in a stable controllable way with minimum waste of sample and maximum hit rate will be tackled with several different development of injector designs and approaches. New developments of the sample delivery system such as liquid mixing jet also opens up new experimental methods which gives opportunities to study of the chemical dynamics in biomolecules in a molecular structural level. The design and characterization of the system will be discussed along with future possible developments and applications. Finally, LCP injector will be discussed which is critical for the success in various applications.
X-Ray crystallography and NMR are two major ways of achieving atomic
resolution of structure determination for macro biomolecules such as proteins. Recently, new developments of hard X-ray pulsed free electron laser XFEL opened up new possibilities to break the dilemma of radiation dose and spatial resolution in diffraction imaging by outrunning radiation damage with ultra high brightness femtosecond X-ray pulses, which is so short in time that the pulse terminates before atomic motion starts. A variety of experimental techniques for structure determination of macro biomolecules is now available including imaging of protein nanocrystals, single particles such as viruses, pump-probe experiments for time-resolved nanocrystallography, and snapshot wide- angle x-ray scattering (WAXS) from molecules in solution. However, due to the nature of the "diffract-then-destroy" process, each protein crystal would be destroyed once
probed. Hence a new sample delivery system is required to replenish the target crystal at a high rate. In this dissertation, the sample delivery systems for the application of XFELs to biomolecular imaging will be discussed and the severe challenges related to the delivering of macroscopic protein crystal in a stable controllable way with minimum waste of sample and maximum hit rate will be tackled with several different development of injector designs and approaches. New developments of the sample delivery system such as liquid mixing jet also opens up new experimental methods which gives opportunities to study of the chemical dynamics in biomolecules in a molecular structural level. The design and characterization of the system will be discussed along with future possible developments and applications. Finally, LCP injector will be discussed which is critical for the success in various applications.
ContributorsWang, Dingjie (Author) / Spence, John CH (Thesis advisor) / Weierstall, Uwe (Committee member) / Schmidt, Kevin (Committee member) / Fromme, Petra (Committee member) / Ozkan, Banu (Committee member) / Arizona State University (Publisher)
Created2014
Description
With the advent of the X-ray free-electron laser (XFEL), an opportunity has arisen to break the nexus between radiation dose and spatial resolution in diffractive imaging, by outrunning radiation damage altogether when using single X-ray pulses so brief that they terminate before atomic motion commences. This dissertation concerns the application of XFELs to biomolecular imaging in an effort to overcome the severe challenges associated with radiation damage and macroscopic protein crystal growth. The method of femtosecond protein nanocrystallography (fsPNX) is investigated, and a new method for extracting crystallographic structure factors is demonstrated on simulated data and on the first experimental fsPNX data obtained at an XFEL. Errors are assessed based on standard metrics familiar to the crystallography community. It is shown that resulting structure factors match the quality of those measured conventionally, at least to 9 angstrom resolution. A new method for ab-initio phasing of coherently-illuminated nanocrystals is then demonstrated on simulated data. The method of correlated fluctuation small-angle X-ray scattering (CFSAXS) is also investigated as an alternative route to biomolecular structure determination, without the use of crystals. It is demonstrated that, for a constrained two-dimensional geometry, a projection image of a single particle can be formed, ab-initio and without modeling parameters, from measured diffracted intensity correlations arising from disordered ensembles of identical particles illuminated simultaneously. The method is demonstrated experimentally, based on soft X-ray diffraction from disordered but identical nanoparticles, providing the first experimental proof-of-principle result. Finally, the fundamental limitations of CFSAXS is investigated through both theory and simulations. It is found that the signal-to-noise ratio (SNR) for CFSAXS data is essentially independent of the number of particles exposed in each diffraction pattern. The dependence of SNR on particle size and resolution is considered, and realistic estimates are made (with the inclusion of solvent scatter) of the SNR for protein solution scattering experiments utilizing an XFEL source.
ContributorsKirian, Richard A (Author) / Spence, John C. H. (Committee member) / Doak, R. Bruce (Committee member) / Weierstall, Uwe (Committee member) / Bennett, Peter (Committee member) / Treacy, Michael M. J. (Committee member) / Arizona State University (Publisher)
Created2011
Description
The properties of materials depend heavily on the spatial distribution and connectivity of their constituent parts. This applies equally to materials such as diamond and glasses as it does to biomolecules that are the product of billions of years of evolution. In science, insight is often gained through simple models with characteristics that are the result of the few features that have purposely been retained. Common to all research within in this thesis is the use of network-based models to describe the properties of materials. This work begins with the description of a technique for decoupling boundary effects from intrinsic properties of nanomaterials that maps the atomic distribution of nanomaterials of diverse shape and size but common atomic geometry onto a universal curve. This is followed by an investigation of correlated density fluctuations in the large length scale limit in amorphous materials through the analysis of large continuous random network models. The difficulty of estimating this limit from finite models is overcome by the development of a technique that uses the variance in the number of atoms in finite subregions to perform the extrapolation to large length scales. The technique is applied to models of amorphous silicon and vitreous silica and compared with results from recent experiments. The latter part this work applies network-based models to biological systems. The first application models force-induced protein unfolding as crack propagation on a constraint network consisting of interactions such as hydrogen bonds that cross-link and stabilize a folded polypeptide chain. Unfolding pathways generated by the model are compared with molecular dynamics simulation and experiment for a diverse set of proteins, demonstrating that the model is able to capture not only native state behavior but also partially unfolded intermediates far from the native state. This study concludes with the extension of the latter model in the development of an efficient algorithm for predicting protein structure through the flexible fitting of atomic models to low-resolution cryo-electron microscopy data. By optimizing the fit to synthetic data through directed sampling and context-dependent constraint removal, predictions are made with accuracies within the expected variability of the native state.
ContributorsDe Graff, Adam (Author) / Thorpe, Michael F. (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Matyushov, Dmitry (Committee member) / Ozkan, Sefika B. (Committee member) / Treacy, Michael M. J. (Committee member) / Arizona State University (Publisher)
Created2011