Matching Items (4)
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- All Subjects: Proteins
- Creators: Redding, Kevin
Description
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is widely accepted as the world's most abundant enzyme and represents the primary entry point for inorganic carbon into the biosphere. Rubisco's slow carboxylation rate of ribulose-1,5-bisphosphate (RuBP) and its susceptibility to inhibition has led some to term it the "bottle neck" of photosynthesis. In order to ensure that Rubisco remains uninhibited, plants require the catalytic chaperone Rubisco activase. Activase is a member of the AAA+ superfamily, ATPases associated with various cellular activities, and uses ATP hydrolysis as the driving force behind a conformational movement that returns activity to inhibited Rubisco active sites. A high resolution activase structure will be an essential tool for examining Rubisco/activase interactions as well as understanding the activase self-association phenomenon. Rubisco activase has long eluded crystallization, likely due to its infamous self-association (polydispersity). Therefore, a limited proteolysis approach was taken to identify soluble activase subdomains as potential crystallization targets. This process involves using proteolytic enzymes to cleave a protein into a few pieces and has previously proven successful in identifying crystallizable protein fragments. Limited proteolysis, utilizing two different proteolytic enzymes (alpha-chymotrypsin and trypsin), identified two tobacco activase products. The fragments that were identified appear to represent most of what is considered to be the AAA+ C-terminal all alpha-domain and some of the AAA+ N-terminal alpha beta alpha-domain. Identified fragments were cloned using the pET151/dTOPO. The project then moved towards cloning and recombinant protein expression in E. coli. NtAbeta(248-383) and NtAbeta(253-354) were successfully cloned, expressed, purified, and characterized through various biophysical techniques. A thermofluor assay of NtAbeta(248-383) revealed a melting temperature of about 30°C, indicating lower thermal stability compared with full-length activase at 43°C. Size exclusion chromatography suggested that NtAbeta(248-383) is monomeric. Circular dichroism was used to identify the secondary structure; a plurality of alpha-helices. NtAbeta(248-383) and NtAbeta(253-354) were subjected to crystallization trials.
ContributorsConrad, Alan (Author) / Wachter, Rebekka (Thesis advisor) / Moore, Thomas (Committee member) / Redding, Kevin (Committee member) / Arizona State University (Publisher)
Created2012
Description
Integrin is a protein in cells that manage cell adhesion. They are crucial to the biochemical functions of cells. L 2 is one type of integrin. Its I domain is responsible for ligand binding. Scientists understand how Alpha L I domain binds Mg2+ at a pH of 7 but not in acidic environments. Knowing the specificity of integrin at a lower pH is important because when tissues become inflamed, they release acidic compounds. We have cloned, expressed, and purified L I-domain and using NMR analysis, we determined that wild type Alpha L I domain does not bind to Mg2+ at a pH of 5.
ContributorsALAM, RAHAT (Author) / Wang, Xu (Thesis director) / Podolnikova, Nataly (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
One of the greatest problems facing society today is the development of a
sustainable, carbon neutral energy source to curb the reliance on fossil fuel combustion as the primary source of energy. To overcome this challenge, research efforts have turned to biology for inspiration, as nature is adept at inter-converting low molecular weight precursors into complex molecules. A number of inorganic catalysts have been reported that mimic the active sites of energy-relevant enzymes such as hydrogenases and carbon monoxide dehydrogenase. However, these inorganic models fail to achieve the high activity of the enzymes, which function in aqueous systems, as they lack the critical secondary-shell interactions that enable the active site of enzymes to outperform their organometallic counterparts.
To address these challenges, my work utilizes bio-hybrid systems in which artificial proteins are used to modulate the properties of organometallic catalysts. This approach couples the diversity of organometallic function with the robust nature of protein biochemistry, aiming to utilize the protein scaffold to not only enhance rates of reaction, but also to control catalytic cycles and reaction outcomes. To this end, I have used chemical biology techniques to modify natural protein structures and augment the H2 producing ability of a cobalt-catalyst by a factor of five through simple mutagenesis. Concurrently I have designed and characterized a de novo peptide that incorporates various iron sulfur clusters at discrete distances from one another, facilitating electron transfer between the two. Finally, using computational methodologies I have engineered proteins to alter the specificity of a CO2 reduction reaction. The proteins systems developed herein allow for study of protein secondary-shell interactions during catalysis, and enable structure-function relationships to be built. The complete system will be interfaced with a solar fuel cell, accepting electrons from a photosensitized dye and storing energy in chemical bonds, such as H2 or methanol.
sustainable, carbon neutral energy source to curb the reliance on fossil fuel combustion as the primary source of energy. To overcome this challenge, research efforts have turned to biology for inspiration, as nature is adept at inter-converting low molecular weight precursors into complex molecules. A number of inorganic catalysts have been reported that mimic the active sites of energy-relevant enzymes such as hydrogenases and carbon monoxide dehydrogenase. However, these inorganic models fail to achieve the high activity of the enzymes, which function in aqueous systems, as they lack the critical secondary-shell interactions that enable the active site of enzymes to outperform their organometallic counterparts.
To address these challenges, my work utilizes bio-hybrid systems in which artificial proteins are used to modulate the properties of organometallic catalysts. This approach couples the diversity of organometallic function with the robust nature of protein biochemistry, aiming to utilize the protein scaffold to not only enhance rates of reaction, but also to control catalytic cycles and reaction outcomes. To this end, I have used chemical biology techniques to modify natural protein structures and augment the H2 producing ability of a cobalt-catalyst by a factor of five through simple mutagenesis. Concurrently I have designed and characterized a de novo peptide that incorporates various iron sulfur clusters at discrete distances from one another, facilitating electron transfer between the two. Finally, using computational methodologies I have engineered proteins to alter the specificity of a CO2 reduction reaction. The proteins systems developed herein allow for study of protein secondary-shell interactions during catalysis, and enable structure-function relationships to be built. The complete system will be interfaced with a solar fuel cell, accepting electrons from a photosensitized dye and storing energy in chemical bonds, such as H2 or methanol.
ContributorsSommer, Dayn (Author) / Ghirlanda, Giovanna (Thesis advisor) / Redding, Kevin (Committee member) / Moore, Gary (Committee member) / Arizona State University (Publisher)
Created2016
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