Matching Items (3)
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- All Subjects: single-molecule
- Creators: Levitus, Marcia
Description
Fluorescence spectroscopy is a popular technique that has been particularly useful in probing biological systems, especially with the invention of single molecule fluorescence. For example, Förster resonance energy transfer (FRET) is one tool that has been helpful in probing distances and conformational changes in biomolecules. In this work, important properties necessary in the quantification of FRET were investigated while FRET was also applied to gain insight into the dynamics of biological molecules. In particular, dynamics of damaged DNA was investigated. While damages in DNA are known to affect DNA structure, what remains unclear is how the presence of a lesion, or multiple lesions, affects the flexibility of DNA, especially in relation to damage recognition by repair enzymes. DNA conformational dynamics was probed by combining FRET and fluorescence anisotropy along with biochemical assays. The focus of this work was to investigate the relationship between dynamics and enzymatic repair. In addition, to properly quantify fluorescence and FRET data, photophysical phenomena of fluorophores, such as blinking, needs to be understood. The triplet formation of the single molecule dye TAMRA and the photoisomerization yield of two different modifications of the single molecule cyanine dye Cy3 were examined spectroscopically to aid in accurate data interpretation. The combination of the biophysical and physiochemical studies illustrates how fluorescence spectroscopy can be used to answer biological questions.
ContributorsShepherd Stennett, Elana Maria (Author) / Levitus, Marcia (Thesis advisor) / Ros, Robert (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2015
Description
Fluorescence spectroscopy is a powerful tool for biophysical studies due to its high sensitivity and broad availability. It is possible to detect fluorescence from single molecules allowing researchers to see the behavior of subpopulations whose presence is obscured by “bulk” collection methods. The fluorescent probes used in these experiments are affected by the solution and macromolecular environments they are in. A misunderstanding of a probe’s photophysics can lead researchers to assign observed behavior to biomolecules, when in fact the probe is responsible. On the other hand, a probe’s photophysical behavior is a signature of the environment surrounding it; it can be exploited to learn about the biomolecule(s) under study. A thorough examination of a probe’s photophysics is critical to data interpretation in both cases and is the focus of this work. This dissertation investigates the photophysical behavior of symmetric and asymmetric cyanines in a variety of solution and biomolecular environments. Using fluorescent techniques—such as time-correlated single photon counting (TCSPC) and fluorescence correlation spectroscopy (FCS)—it was found that cyanines are influenced by the local environment. In the first project, the symmetric cyanines are found to be susceptible to paramagnetic species, such as manganese(II), that enhance the intersystem crossing (ISC) rate increasing triplet blinking and accelerating photobleaching. Another project found the increase in fluorescence of Cy3 in the protein induced fluorescence enhancement (PIFE) technique is due to reduced photoisomerization caused by the proximity of protein to Cy3. The third project focused on asymmetric cyanines; their photophysical behavior has not been previously characterized. Dy630 as a free dye behaves like Cy3; it has a short lifetime and can deactivate via photoisomerization. Preliminary experiments on Dy dyes conjugated to DNA show these dyes do not photoisomerize, and do not show PIFE potential. Further research will explore other conjugation strategies, with the goal of optimizing conditions in which Dy630 can be used as the red-absorbing analogue of Cy3 for PIFE applications. In summary, this dissertation focused on photophysical investigations, the understanding of which forms the backbone of rigorous fluorescent studies and is vital to the development of the fluorescence field.
ContributorsCiuba, Monika A (Author) / Levitus, Marcia (Thesis advisor) / Liu, Yan (Committee member) / Vaiana, Sara (Committee member) / Arizona State University (Publisher)
Created2017
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