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Description
Spinal muscular atrophy (SMA) is a neurodegenerative disease that results in the loss of lower body muscle function. SMA is the second leading genetic cause of death in infants and arises from the loss of the Survival of Motor Neuron (SMN) protein. SMN is produced by two genes, smn1 and

Spinal muscular atrophy (SMA) is a neurodegenerative disease that results in the loss of lower body muscle function. SMA is the second leading genetic cause of death in infants and arises from the loss of the Survival of Motor Neuron (SMN) protein. SMN is produced by two genes, smn1 and smn2, that are identical with the exception of a C to T conversion in exon 7 of the smn2 gene. SMA patients lacking the smn1 gene, rely on smn2 for production of SMN. Due to an alternative splicing event, smn2 primarily encodes a non-functional SMN lacking exon 7 (SMN D7) as well as a low amount of functional full-length SMN (SMN WT). SMN WT is ubiquitously expressed in all cell types, and it remains unclear how low levels of SMN WT in motor neurons lead to motor neuron degradation and SMA. SMN and its associated proteins, Gemin2-8 and Unrip, make up a large dynamic complex that functions to assemble ribonucleoproteins. The aim of this project was to characterize the interactions of the core SMN-Gemin2 complex, and to identify differences between SMN WT and SMN D7. SMN and Gemin2 proteins were expressed, purified and characterized via size exclusion chromatography. A stable N-terminal deleted Gemin2 protein (N45-G2) was characterized. The SMN WT expression system was optimized resulting in a 10-fold increase of protein expression. Lastly, the oligomeric states of SMN and SMN bound to Gemin2 were determined. SMN WT formed a mixture of oligomeric states, while SMN D7 did not. Both SMN WT and D7 bound to Gemin2 with a one-to-one ratio forming a heterodimer and several higher-order oligomeric states. The SMN WT-Gemin2 complex favored high molecular weight oligomers whereas the SMN D7-Gemin2 complex formed low molecular weight oligomers. These results indicate that the SMA mutant protein, SMN D7, was still able to associate with Gemin2, but was not able to form higher-order oligomeric complexes. The observed multiple oligomerization states of SMN and SMN bound to Gemin2 may play a crucial role in regulating one or several functions of the SMN protein. The inability of SMN D7 to form higher-order oligomers may inhibit or alter those functions leading to the SMA disease phenotype.
ContributorsNiday, Tracy (Author) / Allen, James P. (Thesis advisor) / Wachter, Rebekka (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2012
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Description
X-ray diffraction is the technique of choice to determine the three-dimensional structures of proteins. In this study it has been applied to solve the structure of the survival motor neuron (SMN) proteins, the Fenna-Mathews-Olson (FMO) from Pelodictyon phaeum (Pld. phaeum) protein, and the synthetic ATP binding protein DX. Spinal muscular

X-ray diffraction is the technique of choice to determine the three-dimensional structures of proteins. In this study it has been applied to solve the structure of the survival motor neuron (SMN) proteins, the Fenna-Mathews-Olson (FMO) from Pelodictyon phaeum (Pld. phaeum) protein, and the synthetic ATP binding protein DX. Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease resulting in muscle atrophy and paralysis via degeneration of motor neurons in the spinal cord. In this work, we used X-ray diffraction technique to solve the structures of the three variant of the of SMN protein, namely SMN 1-4, SMN-WT, and SMN-Δ7. The SMN 1-4, SMN-WT, and SMN-Δ7 crystals were diffracted to 2.7 Å, 5.5 Å and 3.0 Å, respectively. The three-dimensional structures of the three SMN proteins have been solved. The FMO protein from Pld. phaeum is a water soluble protein that is embedded in the cytoplasmic membrane and serves as an energy transfer funnel between the chlorosome and the reaction center. The FMO crystal diffracted to 1.99Å resolution and the three-dimensional structure has been solved. In previous studies, double mutant, DX, protein was purified and crystallized in the presence of ATP (Simmons et al., 2010; Smith et al. 2007). DX is a synthetic ATP binding protein which resulting from a random selection of DNA library. In this study, DX protein was purified and crystallized without the presence of ATP to investigate the conformational change in DX structure. The crystals of DX were diffracted to 2.5 Å and the three-dimensional structure of DX has been solved.
ContributorsSeng, Chenda O (Author) / Allen, James P. (Thesis advisor) / Wachter, Rebekka (Committee member) / Hayes, Mark (Committee member) / Arizona State University (Publisher)
Created2013
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Description
The utilization of solar energy requires an efficient means of its storage as fuel. In bio-inspired artificial photosynthesis, light energy can be used to drive water oxidation, but catalysts that produce molecular oxygen from water are required. This dissertation demonstrates a novel complex utilizing earth-abundant Ni in combination with glycine

The utilization of solar energy requires an efficient means of its storage as fuel. In bio-inspired artificial photosynthesis, light energy can be used to drive water oxidation, but catalysts that produce molecular oxygen from water are required. This dissertation demonstrates a novel complex utilizing earth-abundant Ni in combination with glycine as an efficient catalyst with a modest overpotential of 0.475 ± 0.005 V for a current density of 1 mA/cm2 at pH 11. The production of molecular oxygen at a high potential was verified by measurement of the change in oxygen concentration, yielding a Faradaic efficiency of 60 ± 5%. This Ni species can achieve a current density of 4 mA/cm2 that persists for at least 10 hours. Based upon the observed pH dependence of the current amplitude and oxidation/reduction peaks, the catalysis is an electron-proton coupled process. In addition, to investigate the binding of divalent metals to proteins, four peptides were designed and synthesized with carboxylate and histidine ligands. The binding of the metals was characterized by monitoring the metal-induced changes in circular dichroism spectra. Cyclic voltammetry demonstrated that bound copper underwent a Cu(I)/Cu(II) oxidation/reduction change at a potential of approximately 0.32 V in a quasi-reversible process. The relative binding affinity of Mn(II), Fe(II), Co(II), Ni(II) and Cu(II) to the peptides is correlated with the stability constants of the Irving-Williams series for divalent metal ions. A potential application of these complexes of transition metals with amino acids or peptides is in the development of artificial photosynthetic cells.
ContributorsWang, Dong (Author) / Allen, James P. (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Redding, Kevin (Committee member) / Arizona State University (Publisher)
Created2014
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Description
The sun provides Earth with a virtually limitless source of energy capable of sustaining all of humanity's needs. Photosynthetic organisms have exploited this energy for eons. However, efficiently converting solar radiation into a readily available and easily transportable form is complex. New materials with optimized physical, electrochemical, and photophysical properties

The sun provides Earth with a virtually limitless source of energy capable of sustaining all of humanity's needs. Photosynthetic organisms have exploited this energy for eons. However, efficiently converting solar radiation into a readily available and easily transportable form is complex. New materials with optimized physical, electrochemical, and photophysical properties are at the forefront of organic solar energy conversion research. In the work presented herein, porphyrin and organometallic dyes with widely-varied properties were studied for solar energy applications. In one project, porphyrins and porphyrin-fullerene dyads with aniline-like features were polymerized via electrochemical methods into semiconductive thin films. These were shown to have high visible light absorption and stable physical and electrochemical properties. However, experimentation using porphyrin polymer films as both the light absorber and semiconductor in a photoelectrochemical cell showed relatively low efficiency of converting absorbed solar energy into electricity. In separate work, tetra-aryl porphyrin derivatives were examined in conjunction with wide-bandgap semiconductive oxides TiO2 and SnO2. Carboxylic acid-, phosphonic acid-, and silatrane-functionalized porphyrins were obtained or synthesized for attachment to the metal oxide species. Electrochemical, photophysical, photoelectrochemical, and surface stability studies of the porphyrins were performed for comparative purposes. The order of surface linkage stability on TiO2 in alkaline conditions, from most stable to least, was determined to be siloxane > phosphonate > carboxylate. Finally, porphyrin dimers fused via their meso and beta positions were synthesized using a chemical oxidative synthesis with a copper(II) oxidant. The molecules exhibit strong absorption in the visible and near-infrared spectral regions as well as interesting electrochemical properties suggesting possible applications in light harvesting and redox catalysis.
ContributorsBrennan, Bradley J (Author) / Gust, Devens (Thesis advisor) / Moore, Thomas A. (Committee member) / Allen, James P. (Committee member) / Arizona State University (Publisher)
Created2012
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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

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