Matching Items (4)
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- All Subjects: Proteins--Analysis.
- All Subjects: BCL2 i-Motif
- Genre: Doctoral Dissertation
- Creators: Levitus, Marcia
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
The communication of genetic material with biomolecules has been a major interest in cancer biology research for decades. Among its different levels of involvement, DNA is known to be a target of several antitumor agents. Additionally, tissue specific interaction between macromolecules such as proteins and structurally important regions of DNA has been reported to define the onset of certain types of cancers.
Illustrated in Chapter 1 is the general history of research on the interaction of DNA and anticancer drugs, most importantly different congener of bleomycin (BLM). Additionally, several synthetic analogues of bleomycin, including the structural components and functionalities, are discussed.
Chapter 2 describes a new approach to study the double-strand DNA lesion caused by antitumor drug bleomycin. The hairpin DNA library used in this study displays numerous cleavage sites demonstrating the versatility of bleomycin interaction with DNA. Interestingly, some of those cleavage sites suggest a novel mechanism of bleomycin interaction, which has not been reported before.
Cytidine methylation has generally been found to decrease site-specific cleavage of DNA by BLM, possibly due to structural change and subsequent reduced bleomycin-mediated recognition of DNA. As illustrated in Chapter 3, three hairpin DNAs known to be strongly bound by bleomycin, and their methylated counterparts, were used to study the dynamics of bleomycin-induced degradation of DNAs in cancer cells. Interestingly, cytidine methylation on one of the DNAs has also shown a major shift in the intensity of bleomycin induced double-strand DNA cleavage pattern, which is known to be a more potent form of bleomycin induced cleavages.
DNA secondary structures are known to play important roles in gene regulation. Chapter 4 demonstrates a structural change of the BCL2 promoter element as a result of its dynamic interaction with the individual domains of hnRNP LL, which is essential to facilitate the transcription of BCL2. Furthermore, an in vitro protein synthesis technique has been employed to study the dynamic interaction between protein domains and the i-motif DNA within the promoter element. Several constructs were made involving replacement of a single amino acid with a fluorescent analogue, and these were used to study FRET between domain 1 and the i-motif, the later of which harbored a fluorescent acceptor nucleotide analogue.
Illustrated in Chapter 1 is the general history of research on the interaction of DNA and anticancer drugs, most importantly different congener of bleomycin (BLM). Additionally, several synthetic analogues of bleomycin, including the structural components and functionalities, are discussed.
Chapter 2 describes a new approach to study the double-strand DNA lesion caused by antitumor drug bleomycin. The hairpin DNA library used in this study displays numerous cleavage sites demonstrating the versatility of bleomycin interaction with DNA. Interestingly, some of those cleavage sites suggest a novel mechanism of bleomycin interaction, which has not been reported before.
Cytidine methylation has generally been found to decrease site-specific cleavage of DNA by BLM, possibly due to structural change and subsequent reduced bleomycin-mediated recognition of DNA. As illustrated in Chapter 3, three hairpin DNAs known to be strongly bound by bleomycin, and their methylated counterparts, were used to study the dynamics of bleomycin-induced degradation of DNAs in cancer cells. Interestingly, cytidine methylation on one of the DNAs has also shown a major shift in the intensity of bleomycin induced double-strand DNA cleavage pattern, which is known to be a more potent form of bleomycin induced cleavages.
DNA secondary structures are known to play important roles in gene regulation. Chapter 4 demonstrates a structural change of the BCL2 promoter element as a result of its dynamic interaction with the individual domains of hnRNP LL, which is essential to facilitate the transcription of BCL2. Furthermore, an in vitro protein synthesis technique has been employed to study the dynamic interaction between protein domains and the i-motif DNA within the promoter element. Several constructs were made involving replacement of a single amino acid with a fluorescent analogue, and these were used to study FRET between domain 1 and the i-motif, the later of which harbored a fluorescent acceptor nucleotide analogue.
ContributorsRoy, Basab (Author) / Hecht, Sidney M. (Thesis advisor) / Jones, Anne (Committee member) / Levitus, Marcia (Committee member) / Chaput, John (Committee member) / Arizona State University (Publisher)
Created2014
Description
Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) has enabled the determination of damage-free protein structures at ambient temperatures and of reaction intermediate species with time resolution on the order of hundreds of femtoseconds. However, currently available XFEL facility X-ray pulse structures waste the majority of continuously injected crystal sample, requiring a large quantity (up to grams) of crystal sample to solve a protein structure. Furthermore, mix-and-inject serial crystallography (MISC) at XFEL facilities requires fast mixing for short (millisecond) reaction time points (𝑡"), and current sample delivery methods have complex fabrication and assembly requirements.
To reduce sample consumption during SFX, a 3D printed T-junction for generating segmented aqueous-in-oil droplets was developed. The device surface properties were characterized both with and without a surface coating for improved droplet generation stability. Additionally, the droplet generation frequency was characterized. The 3D printed device interfaced with gas dynamic virtual nozzles (GDVNs) at the Linac Coherent Light Source (LCLS), and a relationship between the aqueous phase volume and the resulting crystal hit rate was developed. Furthermore, at the European XFEL (EuXFEL) a similar quantity and quality of diffraction data was collected for segmented sample delivery using ~60% less sample volume than continuous injection, and a structure of 3-deoxy-D-manno- octulosonate 8-phosphate synthase (KDO8PS) delivered by segmented injection was solved that revealed new structural details to a resolution of 2.8 Å.
For MISC, a 3D printed hydrodynamic focusing mixer for fast mixing by diffusion was developed to automate device fabrication and simplify device assembly. The mixer was characterized with numerical models and fluorescence microscopy. A variety of devices were developed to reach reaction intermediate time points, 𝑡", on the order of 100 – 103 ms. These devices include 3D printed mixers coupled to glass or 3D printed GDVNs and two designs of mixers with GDVNs integrated into the one device. A 3D printed mixer coupled to a glass GDVN was utilized at LCLS to study the oxidation of cytochrome c oxidase (CcO), and a structure of the CcO Pr intermediate was determined at 𝑡" = 8 s.
To reduce sample consumption during SFX, a 3D printed T-junction for generating segmented aqueous-in-oil droplets was developed. The device surface properties were characterized both with and without a surface coating for improved droplet generation stability. Additionally, the droplet generation frequency was characterized. The 3D printed device interfaced with gas dynamic virtual nozzles (GDVNs) at the Linac Coherent Light Source (LCLS), and a relationship between the aqueous phase volume and the resulting crystal hit rate was developed. Furthermore, at the European XFEL (EuXFEL) a similar quantity and quality of diffraction data was collected for segmented sample delivery using ~60% less sample volume than continuous injection, and a structure of 3-deoxy-D-manno- octulosonate 8-phosphate synthase (KDO8PS) delivered by segmented injection was solved that revealed new structural details to a resolution of 2.8 Å.
For MISC, a 3D printed hydrodynamic focusing mixer for fast mixing by diffusion was developed to automate device fabrication and simplify device assembly. The mixer was characterized with numerical models and fluorescence microscopy. A variety of devices were developed to reach reaction intermediate time points, 𝑡", on the order of 100 – 103 ms. These devices include 3D printed mixers coupled to glass or 3D printed GDVNs and two designs of mixers with GDVNs integrated into the one device. A 3D printed mixer coupled to a glass GDVN was utilized at LCLS to study the oxidation of cytochrome c oxidase (CcO), and a structure of the CcO Pr intermediate was determined at 𝑡" = 8 s.
ContributorsEchelmeier, Austin (Author) / Ros, Alexandra (Thesis advisor) / Levitus, Marcia (Committee member) / Weierstall, Uwe (Committee member) / Arizona State University (Publisher)
Created2019