Matching Items (8)
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- All Subjects: Chemistry, Inorganic
- All Subjects: Lectins
- Creators: Ghirlanda, Giovanna
Description
Cyanovirin-N (CV-N) is a naturally occurring lectin originally isolated from the cyanobacteria Nostoc ellipsosporum. This 11 kDa lectin is 101 amino acids long with two binding sites, one at each end of the protein. CV-N specifically binds to terminal Manα1-2Manα motifs on the branched, high mannose Man9 and Man8 glycosylations found on enveloped viruses including Ebola, Influenza, and HIV. wt-CVN has micromolar binding to soluble Manα1-2Manα and also inhibits HIV entry at low nanomolar concentrations. CV-N's high affinity and specificity for Manα1-2Manα makes it an excellent lectin to study for its glycan-specific properties. The long-term aim of this project is to make a variety of mutant CV-Ns to specifically bind other glycan targets. Such a set of lectins may be used as screening reagents to identify biomarkers and other glycan motifs of interest. As proof of concept, a T7 phage display library was constructed using P51G-m4-CVN genes mutated at positions 41, 44, 52, 53, 56, 74, and 76 in binding Domain B. Five CV-N mutants were selected from the library and expressed in BL21(DE3) E. coli. Two of the mutants, SSDGLQQ-P51Gm4-CVN and AAGRLSK-P51Gm4-CVN, were sufficiently stable for characterization and were examined by CD, Tm, ELISA, and glycan array. Both proteins have CD minima at approximately 213 nm, indicating largely β-sheet structure, and have Tm values greater than 40°C. ELISA against gp120 and RNase B demonstrate both proteins' ability to bind high mannose glycans. To more specifically determine the binding specificity of each protein, AAGRLSK-P51Gm4-CVN, SSDGLQQ-P51Gm4-CVN, wt-CVN, and P51G-m4-CVN were sent to the Consortium for Functional Glycomics (CFG) for glycan array analysis. AAGRLSK-P51Gm4-CVN, wt-CVN, and P51G-m4-CVN, have identical specificities for high mannose glycans containing terminal Manα1-2Manα. SSDGLQQ-P51Gm4-CVN binds to terminal GlcNAcα1-4Gal motifs and a subgroup of high mannose glycans bound by P51G-m4-CVN. SSDGLQQ-wt-CVN was produced to restore anti-HIV activity and has a high nanomolar EC50 value compared to wt-CVN's low nanomolar activity. Overall, these experiments show that CV-N Domain B can be mutated and retain specificity identical to wt-CVN or acquire new glycan specificities. This first generation information can be used to produce glycan-specific lectins for a variety of applications.
ContributorsRuben, Melissa (Author) / Ghirlanda, Giovanna (Thesis advisor) / Allen, James (Committee member) / Wachter, Rebekka (Committee member) / Arizona State University (Publisher)
Created2013
Description
Cyanovirin-N (CVN) is a cyanobacterial lectin with potent anti-HIV activity, mediated by binding to the N-linked oligosaccharide moiety of the envelope protein gp120. CVN offers a scaffold to develop multivalent carbohydrate-binding proteins with tunable specificities and affinities. I present here biophysical calculations completed on a monomeric-stabilized mutant of cyanovirin-N, P51G-m4-CVN, in which domain A binding activity is abolished by four mutations; with comparisons made to CVNmutDB, in which domain B binding activity is abolished. Using Monte Carlo calculations and docking simulations, mutations in CVNmutDB were considered singularly, and the mutations E41A/G and T57A were found to impact the affinity towards dimannose the greatest. 15N-labeled proteins were titrated with Manα(1-2)Manα, while following chemical shift perturbations in NMR spectra. The mutants, E41A/G and T57A, had a larger Kd than P51G-m4-CVN, matching the trends predicted by the calculations. We also observed that the N42A mutation affects the local fold of the binding pocket, thus removing all binding to dimannose. Characterization of the mutant N53S showed similar binding affinity to P51G-m4-CVN. Using biophysical calculations allows us to study future iterations of models to explore affinities and specificities. In order to further elucidate the role of multivalency, I report here a designed covalent dimer of CVN, Nested cyanovirin-N (Nested CVN), which has four binding sites. Nested CVN was found to have comparable binding affinity to gp120 and antiviral activity to wt CVN. These results demonstrate the ability to create a multivalent, covalent dimer that has comparable results to that of wt CVN.
WW domains are small modules consisting of 32-40 amino acids that recognize proline-rich peptides and are found in many signaling pathways. We use WW domain sequences to explore protein folding by simulations using Zipping and Assembly Method. We identified five crucial contacts that enabled us to predict the folding of WW domain sequences based on those contacts. We then designed a folded WW domain peptide from an unfolded WW domain sequence by introducing native contacts at those critical positions.
WW domains are small modules consisting of 32-40 amino acids that recognize proline-rich peptides and are found in many signaling pathways. We use WW domain sequences to explore protein folding by simulations using Zipping and Assembly Method. We identified five crucial contacts that enabled us to predict the folding of WW domain sequences based on those contacts. We then designed a folded WW domain peptide from an unfolded WW domain sequence by introducing native contacts at those critical positions.
ContributorsWoodrum, Brian William (Author) / Ghirlanda, Giovanna (Thesis advisor) / Redding, Kevin (Committee member) / Wang, Xu (Committee member) / Arizona State University (Publisher)
Created2014
Description
Antiviral lectins are potential candidates for future therapies against enveloped viruses like HIV due to their ability to recognize and bind glycans displayed on their surface. Cyanovirin-N (CVN), a lectin that specifically recognizes mannose-rich moieties, serves as a useful model for studying these glycan-recognition mechanisms. This study seeks to improve CVN's glycan-binding affinity by conjugating a boronic acid functional group to the N-terminus via N-terminal specific reductive alkylation by way of a benzaldehyde handle. However, large discrepancies were observed when attempting to confirm a successful conjugation, and further work is necessary to identify the causes and solutions for these issues.
ContributorsDiep, Tristan H (Author) / Ghirlanda, Giovanna (Thesis director) / Redding, Kevin (Committee member) / Mills, Jeremy (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-12
Description
As the return to normality in the wake of the COVID-19 pandemic enters its early stages, the necessity for accurate, quick, and community-wide surveillance of SARS-CoV-2 has been emphasized. Wastewater-based epidemiology (WBE) has been used across the world as a tool for monitoring the pandemic, but studies of its efficacy in comparison to the best-known method for surveillance, randomly selected COVID-19 testing, has limited research. This study evaluated the trends and correlations present between SARS-CoV-2 in the effluent wastewater of a large university campus and random COVID-19 testing results published by the university. A moderately strong positive correlation was found between the random testing and WBE surveillance methods (r = 0.63), and this correlation was strengthened when accommodating for lost samples during the experiment (r = 0.74).
ContributorsWright, Jillian (Author) / Halden, Rolf (Thesis director) / Driver, Erin (Committee member) / School of Music, Dance and Theatre (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
The addition of aminoalkyl-substituted 2,6-bis(imino)pyridine (or pyridine diimine, PDI) ligands to [(COD)RhCl]2 (COD = 1,5-cyclooctadiene) resulted in the formation of rhodium monochloride complexes with the general formula (NPDI)RhCl (NPDI = iPr2NEtPDI or Me2NPrPDI). The investigation of (iPr2NEtPDI)RhCl and (Me2NPrPDI)RhCl by single crystal X-ray diffraction verified the absence of amine arm coordination and a pseudo square planar geometry about rhodium. Replacement of the chloride ligand with an outer-sphere anion was achieved by adding AgBF4 directly to (iPr2NEtPDI)RhCl to form [(iPr2NEtPDI)Rh][BF4]. Alternatively, this complex was prepared upon chelate addition following the salt metathesis reaction between AgBF4 and [(COD)RhCl]2. Using the latter method, both [(NPDI)Rh][BF4] complexes were isolated and found to exhibit κ4-N,N,N,N-PDI coordination regardless of arm length or steric bulk. In contrast, the metallation of PPDI chelates featuring alkylphosphine imine substituents (PPDI = Ph2PEtPDI or Ph2PPrPDI) resulted in the formation of cationic complexes featuring κ5-N,N,N,P,P-PDI coordination in all instances, [(PPDI)Rh][X] (X = Cl, BF4). Adjusting the metallation stoichiometry allowed the preparation of [(Ph2PPrPDI)Rh][(COD)RhCl2], which was characterized by multinuclear NMR spectroscopy and single crystal X-ray diffraction.
ContributorsLevin, Hagit Ben-Daat (Author) / Trovitch, Ryan J (Thesis advisor) / Gould, Ian R (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2016
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
Redox enzymes represent a big group of proteins and they serve as catalysts for
biological processes that involve electron transfer. These proteins contain a redox center
that determines their functional properties, and hence, altering this center or incorporating
non-biological redox cofactor to proteins has been used as a means to generate redox
proteins with desirable activities for biological and chemical applications. Porphyrins and
Fe-S clusters are among the most common cofactors that biology employs for electron
transfer processes and there have been many studies on potential activities that they offer
in redox reactions.
In this dissertation, redox activity of Fe-S clusters and catalytic activity of porphyrins
have been explored with regard to protein scaffolds. In the first part, modular property of
repeat proteins along with previously established protein design principles have been
used to incorporate multiple Fe-S clusters within the repeat protein scaffold. This study is
the first example of exploiting a single scaffold to assemble a determined number of
clusters. In exploring the catalytic activity of transmetallated porphyrins, a cobalt-porphyrin
binding protein known as cytochrome c was employed in a water oxidation
photoelectrochemical cell. This system can be further coupled to a hydrogen production
electrode to achieve a full water splitting tandem cell. Finally, a cobalt-porphyrin binding
protein known as cytochrome b562 was employed to design a whole cell catalysis system,
and the activity of the surface-displayed protein for hydrogen production was explored
photochemically. This system can further be expanded for directed evolution studies and
high-throughput screening.
biological processes that involve electron transfer. These proteins contain a redox center
that determines their functional properties, and hence, altering this center or incorporating
non-biological redox cofactor to proteins has been used as a means to generate redox
proteins with desirable activities for biological and chemical applications. Porphyrins and
Fe-S clusters are among the most common cofactors that biology employs for electron
transfer processes and there have been many studies on potential activities that they offer
in redox reactions.
In this dissertation, redox activity of Fe-S clusters and catalytic activity of porphyrins
have been explored with regard to protein scaffolds. In the first part, modular property of
repeat proteins along with previously established protein design principles have been
used to incorporate multiple Fe-S clusters within the repeat protein scaffold. This study is
the first example of exploiting a single scaffold to assemble a determined number of
clusters. In exploring the catalytic activity of transmetallated porphyrins, a cobalt-porphyrin
binding protein known as cytochrome c was employed in a water oxidation
photoelectrochemical cell. This system can be further coupled to a hydrogen production
electrode to achieve a full water splitting tandem cell. Finally, a cobalt-porphyrin binding
protein known as cytochrome b562 was employed to design a whole cell catalysis system,
and the activity of the surface-displayed protein for hydrogen production was explored
photochemically. This system can further be expanded for directed evolution studies and
high-throughput screening.
ContributorsBahrami Dizicheh, Zahra (Author) / Ghirlanda, Giovanna (Thesis advisor) / Allen, James P. (Committee member) / Seo, Dong Kyun (Committee member) / Arizona State University (Publisher)
Created2019
Description
Coronavirus disease 2019 (COVID-19), an illness caused by severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2), has been responsible for significant social and economic
disruption, prompting an urgent search for therapeutic solutions. The spike protein of the virus
has been examined as an immunogenic target because of its role in viral binding and fusion
necessary for infection of host cells. Previous studies have identified a recombinant protein
(denoted as S1) that has been shown to potentially induce a neutralizing antibody response by
mimicking the structure of the SARS-CoV-2 spike protein. We have produced the S1 in plants
using agroinfiltration, a plant transformation technique whereby plasmid-containing
Agrobacterium tumefaciens is injected into Nicotiana benthamiana plants, resulting in transfer of
the desired gene from bacteria to plant cells. S1 was expressed to high levels within 5 days of
infiltration, and Western blot analysis showed recognition of the S1 by an anti-S1 antibody.
ELISA results exhibited increased binding activity to anti-S1 with increasing concentrations of
S1, indicating their specific interaction. This ongoing study will demonstrate the potential of a
plant-produced S1 as a vaccine, therapeutic, and diagnostic tool against COVID-19 that is not
only effective, but also cost-efficient and scalable in comparison to conventional mammalian cell
culture production methods.
coronavirus 2 (SARS-CoV-2), has been responsible for significant social and economic
disruption, prompting an urgent search for therapeutic solutions. The spike protein of the virus
has been examined as an immunogenic target because of its role in viral binding and fusion
necessary for infection of host cells. Previous studies have identified a recombinant protein
(denoted as S1) that has been shown to potentially induce a neutralizing antibody response by
mimicking the structure of the SARS-CoV-2 spike protein. We have produced the S1 in plants
using agroinfiltration, a plant transformation technique whereby plasmid-containing
Agrobacterium tumefaciens is injected into Nicotiana benthamiana plants, resulting in transfer of
the desired gene from bacteria to plant cells. S1 was expressed to high levels within 5 days of
infiltration, and Western blot analysis showed recognition of the S1 by an anti-S1 antibody.
ELISA results exhibited increased binding activity to anti-S1 with increasing concentrations of
S1, indicating their specific interaction. This ongoing study will demonstrate the potential of a
plant-produced S1 as a vaccine, therapeutic, and diagnostic tool against COVID-19 that is not
only effective, but also cost-efficient and scalable in comparison to conventional mammalian cell
culture production methods.
ContributorsNguyen, Katherine (Author) / Chen, Qiang (Thesis director) / Ghirlanda, Giovanna (Committee member) / Jugler, Collin (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-12