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- Creators: Barrett, The Honors College
- Creators: Fromme, Petra
- Creators: Levitus, Marcia
Description
Membrane proteins are very important for all living cells, being involved in respiration, photosynthesis, cellular uptake and signal transduction, amongst other vital functions. However, less than 300 unique membrane protein structures have been determined to date, often due to difficulties associated with the growth of sufficiently large and well-ordered crystals. This work has been focused on showing the first proof of concept for using membrane protein nanocrystals and microcrystals for high-resolution structure determination. Upon determining that crystals of the membrane protein Photosystem I, which is the largest and most complex membrane protein crystallized to date, exist with only a hundred unit cells with sizes of less than 200 nm on an edge, work was done to develop a technique that could exploit the growth of the Photosystem I nanocrystals and microcrystals. Femtosecond X-ray protein nanocrystallography was developed for use at the first high-energy X-ray free electron laser, the LCLS at SLAC National Accelerator Laboratory, in which a liquid jet would bring fully hydrated Photosystem I nanocrystals into the interaction region of the pulsed X-ray source. Diffraction patterns were recorded from millions of individual PSI nanocrystals and data from thousands of different, randomly oriented crystallites were integrated using Monte Carlo integration of the peak intensities. The short pulses ( 70 fs) provided by the LCLS allowed the possibility to collect the diffraction data before the onset of radiation damage, exploiting the diffract-before-destroy principle. At the initial experiments at the AMO beamline using 6.9- Å wavelength, Bragg peaks were recorded to 8.5- Å resolution, and an electron-density map was determined that did not show any effects of X-ray-induced radiation damage. Recently, femtosecond X-ray protein nanocrystallography experiments were done at the CXI beamline of the LCLS using 1.3- Å wavelength, and Bragg reflections were recorded to 3- Å resolution; the data are currently being processed. Many additional techniques still need to be developed to explore the femtosecond nanocrystallography technique for experimental phasing and time-resolved X-ray crystallography experiments. The first proof-of-principle results for the femtosecond nanocrystallography technique indicate the incredible potential of the technique to offer a new route to the structure determination of membrane proteins.
ContributorsHunter, Mark (Author) / Fromme, Petra (Thesis advisor) / Wolf, George (Committee member) / Levitus, Marcia (Committee member) / Arizona State University (Publisher)
Created2011
Description
ATP synthase is a large multimeric protein complex responsible for generating the energy molecule adenosine triphosphate (ATP) in most organisms. The catalysis involves the rotation of a ring of c-subunits, which is driven by the transmembrane electrochemical gradient. This dissertation reports how the eukaryotic c-subunit from spinach chloroplast ATP synthase has successfully been expressed in Escherichia coli and purified in mg quantities by incorporating a unique combination of methods. Expression was accomplished using a codon optimized gene for the c-subunit, and it was expressed as an attachment to the larger, more soluble, native maltose binding protein (MBP-c1). The fusion protein MBP-c1 was purified on an affinity column, and the c1 subunit was subsequently severed by protease cleavage in the presence of detergent. Final purification of the monomeric c1 subunit was accomplished using reversed phase column chromatography with ethanol as an eluent. Circular dichroism spectroscopy data showed clear evidence that the purified c-subunit is folded with the native alpha-helical secondary structure. Recent experiments appear to indicate that this monomeric recombinant c-subunit forms an oligomeric ring that is similar to its native tetradecameric form when reconstituted in liposomes. The F-type ATP synthase c-subunit stoichiometry is currently known to vary from 8 to 15 subunits among different organisms. This has a direct influence on the metabolic requirements of the corresponding organism because each c-subunit binds and transports one H+ across the membrane as the ring makes a complete rotation. The c-ring rotation drives rotation of the gamma-subunit, which in turn drives the synthesis of 3 ATP for every complete rotation. The availability of a recombinantly produced c-ring will lead to new experiments which can be designed to investigate the possible factors that determine the variable c-ring stoichiometry and structure.
ContributorsLawrence, Robert Michael (Author) / Fromme, Petra (Thesis advisor) / Chen, Julian J.L. (Committee member) / Woodbury, Neal W. (Committee member) / Arizona State University (Publisher)
Created2011
Description
Conformational changes in biomolecules often take place on longer timescales than are easily accessible with unbiased molecular dynamics simulations, necessitating the use of enhanced sampling techniques, such as adaptive umbrella sampling. In this technique, the conformational free energy is calculated in terms of a designated set of reaction coordinates. At the same time, estimates of this free energy are subtracted from the potential energy in order to remove free energy barriers and cause conformational changes to take place more rapidly. This dissertation presents applications of adaptive umbrella sampling to a variety of biomolecular systems. The first study investigated the effects of glycosylation in GalNAc2-MM1, an analog of glycosylated macrophage activating factor. It was found that glycosylation destabilizes the protein by increasing the solvent exposure of hydrophobic residues. The second study examined the role of bound calcium ions in promoting the isomerization of a cis peptide bond in the collagen-binding domain of Clostridium histolyticum collagenase. This study determined that the bound calcium ions reduced the barrier to the isomerization of this peptide bond as well as stabilizing the cis conformation thermodynamically, and identified some of the reasons for this. The third study represents the application of GAMUS (Gaussian mixture adaptive umbrella sampling) to on the conformational dynamics of the fluorescent dye Cy3 attached to the 5' end of DNA, and made predictions concerning the affinity of Cy3 for different base pairs, which were subsequently verified experimentally. Finally, the adaptive umbrella sampling method is extended to make use of the roll angle between adjacent base pairs as a reaction coordinate in order to examine the bending both of free DNA and of DNA bound to the archaeal protein Sac7d. It is found that when DNA bends significantly, cations from the surrounding solution congregate on the concave side, which increases the flexibility of the DNA by screening the repulsion between phosphate backbones. The flexibility of DNA on short length scales is compared to the worm-like chain model, and the contribution of cooperativity in DNA bending to protein-DNA binding is assessed.
ContributorsSpiriti, Justin Matthew (Author) / van der Vaart, Arjan (Thesis advisor) / Chizmeshya, Andrew (Thesis advisor) / Matyushov, Dmitry (Committee member) / Fromme, Petra (Committee member) / Arizona State University (Publisher)
Created2011
Description
This dissertation describes the work on two projects which involves measuring molecular conductance and studying their properties on the nanoscale using various Scanning Tunneling Microscopy (STM) techniques. The first molecule studied was a porphyrin-fullerene moiety known as a molecular Dyad for photovoltaic applications. This project is further divided into two section, the first one involving the characterization of the Dyad monolayers and conductance measurement in the dark. The Dyads are designed to form charge separated states on illumination. The lifetime of the charged states have been measured efficiently but the single-molecule conductance through the molecules have yet to be characterized. The second part of the project describes the set-up of a novel sample stage which enables the study of molecular conductance under illumination. This part also describes the subsequent study of the molecule under illumination and the observation of a unique charge-separated state. It also contains the verification of the presence of this charge-separated using other characterization techniques like transient absorption spectroscopy. The second project described in the dissertation was studying and comparing the predicted rectifying nature of two molecules, identical in every way except for one stereocenter. This project describes the formation of monolayers of the molecule on gold and then studying and analyzing the current-voltage characteristics of the molecules and looking for rectification. Both the molecules proved to be rectifying, one more than the other as predicted by theoretical calculations.
ContributorsBhattacharyya, Shreya (Author) / Lindsay, Stuart (Thesis advisor) / Moore, Ana (Committee member) / Levitus, Marcia (Committee member) / Arizona State University (Publisher)
Created2011
Description
Solution conformations and dynamics of proteins and protein-DNA complexes are often difficult to predict from their crystal structures. The crystal structure only shows a snapshot of the different conformations these biological molecules can have in solution. Multiple different conformations can exist in solution and potentially have more importance in the biological activity. DNA sliding clamps are a family of proteins with known crystal structures. These clamps encircle the DNA and enable other proteins to interact more efficiently with the DNA. Eukaryotic PCNA and prokaryotic β clamp are two of these clamps, some of the most stable homo-oligomers known. However, their solution stability and conformational equilibrium have not been investigated in depth before. Presented here are the studies involving two sliding clamps: yeast PCNA and bacterial β clamp. These studies show that the β clamp has a very different solution stability than PCNA. These conclusions were reached through various different fluorescence-based experiments, including fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), single molecule fluorescence, and various time resolved fluorescence techniques. Interpretations of these, and all other, fluorescence-based experiments are often affected by the properties of the fluorophores employed. Often the fluorescence properties of these fluorophores are influenced by their microenvironments. Fluorophores are known to sometimes interact with biological molecules, and this can have pronounced effects on the rotational mobility and photophysical properties of the dye. Misunderstanding the effect of these photophysical and rotational properties can lead to a misinterpretation of the obtained data. In this thesis, photophysical behaviors of various organic dyes were studied in the presence of deoxymononucleotides to examine more closely how interactions between fluorophores and DNA bases can affect fluorescent properties. Furthermore, the properties of cyanine dyes when bound to DNA and the effect of restricted rotation on FRET are presented in this thesis. This thesis involves studying fluorophore photophysics in various microenvironments and then expanding into the solution stability and dynamics of the DNA sliding clamps.
ContributorsRanjit, Suman (Author) / Levitus, Marcia (Thesis advisor) / Lindsay, Stuart (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2013
Description
Single molecule DNA Sequencing technology has been a hot research topic in the recent decades because it holds the promise to sequence a human genome in a fast and affordable way, which will eventually make personalized medicine possible. Single molecule differentiation and DNA translocation control are the two main challenges in all single molecule DNA sequencing methods. In this thesis, I will first introduce DNA sequencing technology development and its application, and then explain the performance and limitation of prior art in detail. Following that, I will show a single molecule DNA base differentiation result obtained in recognition tunneling experiments. Furthermore, I will explain the assembly of a nanofluidic platform for single strand DNA translocation, which holds the promised to be integrated into a single molecule DNA sequencing instrument for DNA translocation control. Taken together, my dissertation research demonstrated the potential of using recognition tunneling techniques to serve as a general readout system for single molecule DNA sequencing application.
ContributorsLiu, Hao (Author) / Lindsay, Stuart M (Committee member) / Yan, Hao (Committee member) / Levitus, Marcia (Committee member) / Arizona State University (Publisher)
Created2013
Description
The heliobacterial reaction center (HbRC) is widely considered the simplest and most primitive photosynthetic reaction center (RC) still in existence. Despite the simplicity of the HbRC, many aspects of the electron transfer mechanism remain unknown or under debate. Improving our understanding of the structure and function of the HbRC is important in determining its role in the evolution of photosynthetic RCs. In this work, the function and properties of the iron-sulfur cluster FX and quinones of the HbRC were investigated, as these are the characteristic terminal electron acceptors used by Type-I and Type-II RCs, respectively. In Chapter 3, I develop a system to directly detect quinone double reduction activity using reverse-phase high pressure liquid chromatography (RP-HPLC), showing that Photosystem I (PSI) can reduce PQ to PQH2. In Chapter 4, I use RP-HPLC to characterize the HbRC, showing a surprisingly small antenna size and confirming the presence of menaquinone (MQ) in the isolated HbRC. The terminal electron acceptor FX was characterized spectroscopically and electrochemically in Chapter 5. I used three new systems to reduce FX in the HbRC, using EPR to confirm a S=3/2 ground-state for the reduced cluster. The midpoint potential of FX determined through thin film voltammetry was -372 mV, showing the cluster is much less reducing than previously expected. In Chapter 7, I show light-driven reduction of menaquinone in heliobacterial membrane samples using only mild chemical reductants. Finally, I discuss the evolutionary implications of these findings in Chapter 7.
ContributorsCowgill, John (Author) / Redding, Kevin (Thesis advisor) / Jones, Anne (Committee member) / Fromme, Petra (Committee member) / Arizona State University (Publisher)
Created2012
Description
Lyme disease is a common tick-borne illness caused by the Gram-negative bacterium Borrelia burgdorferi. An outer membrane protein of Borrelia burgdorferi, P66, has been suggested as a possible target for Lyme disease treatments. However, a lack of structural information available for P66 has hindered attempts to design medications to target the protein. Therefore, this study attempted to find methods for expressing and purifying P66 in quantities that can be used for structural studies. It was found that by using the PelB signal sequence, His-tagged P66 could be directed to the outer membrane of Escherichia coli, as confirmed by an anti-His Western blot. Further attempts to optimize P66 expression in the outer membrane were made, pending verification via Western blotting. The ability to direct P66 to the outer membrane using the PelB signal sequence is a promising first step in determining the overall structure of P66, but further work is needed before P66 is ready for large-scale purification for structural studies.
ContributorsRamirez, Christopher Nicholas (Author) / Fromme, Petra (Thesis director) / Hansen, Debra (Committee member) / Department of Physics (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
One major issue that surgeons face during closed body cavity surgery is fogging of the lens surfaces. The cloudy and opaque lens surface caused by water vapor present in closed body cavities forces the surgeon to repeatedly remove the endoscope, wipe it, and reinsert it back into the patient. This presents several risks such as increased surgery time, greater scarring, and an increased chance of infection. In order to address this issue, the development of the Thin Fluid Film Device (TFFD™) VitreOx™ aims to render the lens surface hydrophilic, whereas it is typically hydrophobic. By creating a hydrophilic polymeric nanomesh, the 3-D water droplets can be trapped to lie flatter, thus resulting in a flatter 2-D sheeting effect. The light can no longer be refracted at different angles off of the 3-dimensional water beads, thus eliminating the opacity of the lens surface.
Two animal trials were performed involving a rat and two pigs in order to prove the efficacy of VitreOx™ in addition to being compared with competitor, Covidien Clearify. A laparoscopy was performed on each animal, and the length of time that the endoscope took to fog was measured post product application. The results of the optimized animal clinical trials involving two Yucatan pigs showed that the scope treated with Covidien’s Clearify began fogging within 8 minutes and continued to do so for the remained of the surgery, as opposed to the scope with VitreOx™ which remained fog free for the full 90-minute procedure. The results proved the efficacy of our product.
The second part of the thesis aimed to optimize HemoClear™, the blood evacuating TFFD™. This was done by testing a higher concentration of 6 mg/mL fibrinogen as compared to previous work. After conducting an experiment designed to mimic closed-body cavity surgery it was determined that the HemoClear™ eliminated fog 67% of the time and evacuated blood with a success of 83%. Future work aims to continue testing at this concentration with variances in mixing and application technique.
Two animal trials were performed involving a rat and two pigs in order to prove the efficacy of VitreOx™ in addition to being compared with competitor, Covidien Clearify. A laparoscopy was performed on each animal, and the length of time that the endoscope took to fog was measured post product application. The results of the optimized animal clinical trials involving two Yucatan pigs showed that the scope treated with Covidien’s Clearify began fogging within 8 minutes and continued to do so for the remained of the surgery, as opposed to the scope with VitreOx™ which remained fog free for the full 90-minute procedure. The results proved the efficacy of our product.
The second part of the thesis aimed to optimize HemoClear™, the blood evacuating TFFD™. This was done by testing a higher concentration of 6 mg/mL fibrinogen as compared to previous work. After conducting an experiment designed to mimic closed-body cavity surgery it was determined that the HemoClear™ eliminated fog 67% of the time and evacuated blood with a success of 83%. Future work aims to continue testing at this concentration with variances in mixing and application technique.
ContributorsSinha, Saloni Agarwal (Author) / Culbertson, Robert (Thesis director) / Herbots, Nicole (Committee member) / Watson, Clarizza (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
Exploring Structure and Function of Human Cold Sensing Protein TRPM8 with ROSETTA Comparative Models
Description
Transient Receptor Potential (TRP) channels are a diverse class of ion channels notable as polymodal sensors. TRPM8 is a TRP channel implicated in cold sensation, nociception, and a variety of human diseases, including obesity and cancer. Despite sustained interest in TRPM8 since its discovery in 2001, many of the molecular mechanisms that underlie function are not yet clear. Knowledge of these properties could have implications for medicine and physiological understanding of sensation and signaling. Structures of TRP channels have proven challenging to solve, but recent Cryoelectron microscopy (Cryo-EM) structures of TRPV1 provide a basis for homology-based modeling of TRP channel structures and interactions. I present an ensemble of 11,000 Rosetta computational homology models of TRPM8 based on the recent Cryo-EM apo structure of TRPV1 (PDB code:3J5P). Site-directed mutagenesis has provided clues about which residues are most essential for modulatory ligands to bind, so the models presented provide a platform to investigate the structural basis of TRPM8 ligand modulation complementary to existing functional and structural information. Menthol and icilin appear to interact with interfacial residues in the sensor domain (S1-S4). One consensus feature of these sites is the presence of local contacts to the S4 helix, suggesting this helix may be mechanistically involved with the opening of the pore. Phosphatidylinositol 4,5-bisphosphate (PIP2)has long been known to interact with the C-terminus of TRPM8, and some of the homology models contain plausible binding pockets where PIP2 can come into contact with charged residues known to be essential for PIP2 modulation. Future in silico binding experiments could provide testable hypothesis for in vitro structural studies, and experimental data (e.g. distance constraints from electron paramagnetic resonance spectroscopy [EPR]) could further refine the models.
ContributorsHelsell, Cole Vincent Maher (Author) / Van Horn, Wade (Thesis director) / Wang, Xu (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05