Matching Items (13)

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Dependence of the angular velocity of rotation on rotational position at which ATP-binding occurs at the empty catalytic site of the F1-ATPase molecular motor

Description

The FoF1 ATP synthase is a molecular motor critical to the metabolism of virtually all life forms, and it acts in the manner of a hydroelectric generator. The F1 complex

The FoF1 ATP synthase is a molecular motor critical to the metabolism of virtually all life forms, and it acts in the manner of a hydroelectric generator. The F1 complex contains an (αβ)3 (hexamer) ring in which catalysis occurs, as well as a rotor comprised by subunit-ε in addition to the coiled-coil and globular foot domains of subunit-γ. The F1 complex can hydrolyze ATP in vitro in a manner that drives counterclockwise (CCW) rotation, in 120° power strokes, as viewed from the positive side of the membrane. The power strokes that occur in ≈ 300 μsec are separated by catalytic dwells that occur on a msec time scale. A single-molecule rotation assay that uses the intensity of polarized light, scattered from a 75 × 35 nm gold nanorod, determined the average rotational velocity of the power stroke (ω, in degrees/ms) as a function of the rotational position of the rotor (θ, in degrees, measured in reference to the catalytic dwell). The velocity is not constant but rather accelerates and decelerates in two Phases. Phase-1 (0° - 60°) is believed to derive power from elastic energy in the protein. At concentrations of ATP that limit the rate of ATP hydrolysis, the rotor can stop for an ATP-binding dwell during Phase-1. Although the most probable position that the ATP-binding dwell occurs is 40° after the catalytic dwell, the ATP-binding dwell can occur at any rotational position during Phase-1 of the power stroke. Phase-2 of the power stroke (60° - 120°) is believed to be powered by the ATP-binding induced closure of the lever domain of a β-subunit (as it acts as a cam shaft against the γ-subunit). Algorithms were written, to sort and analyze F1-ATPase power strokes, to determine the average rotational velocity profile of power strokes as a function of the rotational position at which the ATP-binding dwell occurs (θATP-bd), and when the ATP-binding dwell is absent. Sorting individual ω(θ) curves, as a function of θATP-bd, revealed that a dependence of ω on
θATP-bd exists. The ATP-binding dwell can occur even at saturating ATP concentrations. We report that ω follows a distinct pattern in the vicinity of the ATP-binding dwell, and that the ω(θ) curve contains the same oscillations within it regardless of θATP-bd. We observed that an acceleration/deceleration dependence before and after the ATP-binding dwell, respectively, remained for increasing time intervals as the dwell occurred later in Phase-1, to a maximum of ≈ 40°. The results were interpreted in terms of a model in which the ATP-binding dwell results from internal drag at a variable position on the γε rotor.

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  • 2016-12

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Metal Replacement Studies in Bacillus subtilis Quercetinase

Description

Quercetin 2,3-dioxygenase from Bacillus subtilis has been identified and characterized as the first known prokaryotic quercetinase. This enzyme catalyzes the cleavage of the O-heteroaromatic ring of the flavonol quercetin to

Quercetin 2,3-dioxygenase from Bacillus subtilis has been identified and characterized as the first known prokaryotic quercetinase. This enzyme catalyzes the cleavage of the O-heteroaromatic ring of the flavonol quercetin to the corresponding depside and carbon monoxide. The first quercetinase was characterized from a species of Aspergillus genus, and was found to contain one Cu2+ per subunit. For many years, it was thought that the B. subtilis quercetinase contained two Fe2+ ions per subunit; however, it has since been discovered that Mn2+ is a much more likely cofactor. Studies of overexpressed bacterial enzyme in E. coli indicated that this enzyme may be active with other metal ions (e.g. Co2+); however, the production of enzyme with full metal incorporation has only been possible with Mn2+. This study explores the notion that metal manipulation after translation, by partially unfolding the enzyme, chelating the metal ions, and then refolding the protein in the presence of an excess of divalent metal ions, could generate enzyme with full metal occupancy. The protocols presented here included testing for activity after incubating purified quercetinase with EDTA, DDTC, imidazole and GndHCl. It was found that the metal chelators had little to no effect on quercetinase activity. Imidazole did appear to inhibit the enzyme at concentrations in the millimolar range. In addition, the quercetinase was denatured in GndHCl at concentrations above 1 M. Recovering an active enzyme after partial or complete unfolding proved difficult, if not impossible.

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  • 2014-05

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Studies on the three-dimensional structures of proteins using X-ray crystallography

Description

X-ray diffraction is the technique of choice to determine the three-dimensional structures of proteins. In this study it has been applied to solve the structure of the survival motor neuron

X-ray diffraction is the technique of choice to determine the three-dimensional structures of proteins. In this study it has been applied to solve the structure of the survival motor neuron (SMN) proteins, the Fenna-Mathews-Olson (FMO) from Pelodictyon phaeum (Pld. phaeum) protein, and the synthetic ATP binding protein DX. Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease resulting in muscle atrophy and paralysis via degeneration of motor neurons in the spinal cord. In this work, we used X-ray diffraction technique to solve the structures of the three variant of the of SMN protein, namely SMN 1-4, SMN-WT, and SMN-Δ7. The SMN 1-4, SMN-WT, and SMN-Δ7 crystals were diffracted to 2.7 Å, 5.5 Å and 3.0 Å, respectively. The three-dimensional structures of the three SMN proteins have been solved. The FMO protein from Pld. phaeum is a water soluble protein that is embedded in the cytoplasmic membrane and serves as an energy transfer funnel between the chlorosome and the reaction center. The FMO crystal diffracted to 1.99Å resolution and the three-dimensional structure has been solved. In previous studies, double mutant, DX, protein was purified and crystallized in the presence of ATP (Simmons et al., 2010; Smith et al. 2007). DX is a synthetic ATP binding protein which resulting from a random selection of DNA library. In this study, DX protein was purified and crystallized without the presence of ATP to investigate the conformational change in DX structure. The crystals of DX were diffracted to 2.5 Å and the three-dimensional structure of DX has been solved.

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Date Created
  • 2013

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Synthesis and characterization of dyes with solar energy applications

Description

The sun provides Earth with a virtually limitless source of energy capable of sustaining all of humanity's needs. Photosynthetic organisms have exploited this energy for eons. However, efficiently converting solar

The sun provides Earth with a virtually limitless source of energy capable of sustaining all of humanity's needs. Photosynthetic organisms have exploited this energy for eons. However, efficiently converting solar radiation into a readily available and easily transportable form is complex. New materials with optimized physical, electrochemical, and photophysical properties are at the forefront of organic solar energy conversion research. In the work presented herein, porphyrin and organometallic dyes with widely-varied properties were studied for solar energy applications. In one project, porphyrins and porphyrin-fullerene dyads with aniline-like features were polymerized via electrochemical methods into semiconductive thin films. These were shown to have high visible light absorption and stable physical and electrochemical properties. However, experimentation using porphyrin polymer films as both the light absorber and semiconductor in a photoelectrochemical cell showed relatively low efficiency of converting absorbed solar energy into electricity. In separate work, tetra-aryl porphyrin derivatives were examined in conjunction with wide-bandgap semiconductive oxides TiO2 and SnO2. Carboxylic acid-, phosphonic acid-, and silatrane-functionalized porphyrins were obtained or synthesized for attachment to the metal oxide species. Electrochemical, photophysical, photoelectrochemical, and surface stability studies of the porphyrins were performed for comparative purposes. The order of surface linkage stability on TiO2 in alkaline conditions, from most stable to least, was determined to be siloxane > phosphonate > carboxylate. Finally, porphyrin dimers fused via their meso and beta positions were synthesized using a chemical oxidative synthesis with a copper(II) oxidant. The molecules exhibit strong absorption in the visible and near-infrared spectral regions as well as interesting electrochemical properties suggesting possible applications in light harvesting and redox catalysis.

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Date Created
  • 2012

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Structural and functional interrogation of single amino acid residues in fluorescent proteins

Description

Acquisition of fluorescence via autocatalytic processes is unique to few proteins in the natural world. Fluorescent proteins (FPs) have been integral to live-cell imaging techniques for decades; however, mechanistic information

Acquisition of fluorescence via autocatalytic processes is unique to few proteins in the natural world. Fluorescent proteins (FPs) have been integral to live-cell imaging techniques for decades; however, mechanistic information is still emerging fifty years after the discovery of the original green fluorescent protein (GFP). Modification of the fluorescence properties of the proteins derived from GFP allows increased complexity of experiments and consequently, information content of the data acquired. The importance of arginine-96 in GFP has been widely discussed. It has been established as vital to the kinetics of chromophore maturation and to the overall fold of GFP before post-translational self-modification. Its value during chromophore maturation has been demonstrated by mutational studies and a hypothesis proposed for its catalytic function. A strategy is described herein to determine its pKa value via NMR to determine whether Arg96 possesses the chemical capacity to function as a general base during GFP chromophore biosynthesis. Förster resonance energy transfer (FRET) techniques commonly employ Enhanced Cyan Fluorescent Proteins (ECFPs) and their derivatives as donor fluorophores useful in real-time, live-cell imaging. These proteins have a tryptophan-derived chromophore that emits light in the blue region of the visible spectrum. Most ECFPs suffer from fluorescence instability, which, coupled with their low quantum yield, makes data analysis unreliable. The structural heterogeneity of these proteins also results in undesirable photophysical characteristics. Recently, mCerulean3, a ten amino acid mutant of ECFP, was introduced as an optimized FRET-donor protein (1). The amino acids changed include a mobile residue, Asp148, which has been mutated to a glycine in the new construct, and Thr65 near the chromophore has been mutated to a serine, the wild-type residue at this location. I have solved the x-ray crystal structure of mCerulean3 at low pH and find that the pH-dependent isomerization has been eliminated. The chromophore is in the trans-conformation previously observed in Cerulean at pH 8. The mutations that increase the quantum yield and improve fluorescence brightness result in a stable, bright donor fluorophore well-suited for use in quantitative microscopic imaging.

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Date Created
  • 2012

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Design of Redox Proteins as Catalysts for Fuel Production

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

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.

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Created

Date Created
  • 2019

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Characterization of Intrinsic and Extrinsic Factors that Regulate Human Telomerase Repeat Addition

Description

The linear chromosomes ends in eukaryotes are protected by telomeres, a nucleoprotein structure that contains telomeric DNA with repetitive sequence and associated proteins. Telomerase is an RNA-dependent DNA polymerase that

The linear chromosomes ends in eukaryotes are protected by telomeres, a nucleoprotein structure that contains telomeric DNA with repetitive sequence and associated proteins. Telomerase is an RNA-dependent DNA polymerase that adds telomeric DNA repeats to the 3'-ends of chromosomes to offset the loss of terminal DNA repeats during DNA replication. It consists of two core components: a telomerase reverse transcriptase (TERT) and a telomerase RNA (TR). Telomerase uses a short sequence in its integral RNA component as template to add multiple DNA repeats in a processive manner. However, it remains unclear how the telomerase utilizes the short RNA template accurately and efficiently during DNA repeat synthesis. As previously reported human telomerase nucleotide synthesis arrests upon reaching the end of its RNA template by a unique template-embedded pause signal. In this study, I demonstrate pause signal remains active following template regeneration and inhibits the intrinsic processivity and rate of telomerase repeat addition. Furthermore, I have found that the human telomerase catalytic cycle comprises a crucial and slow incorporation of the first nucleotide after template translocation. This slow nucleotide incorporation step drastically limits repeat addition processivity and rate, which is alleviated with elevated concentrations of dGTP. Additionally, molecular mechanism of the disease mutants on telomerase specific motif T, K570N, have been explored. Finally, I studied how telomerase selective inhibitor BIBR 1532 reduce telomerase repeat addition processivity by function assay. Together, these results shed new light on telomerase catalytic cycle and the importance of telomerase for biomedicine.

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Date Created
  • 2018

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Structure and function of the homodimeric reaction center, and hydrogen production, in Heliobacterium modesticaldum

Description

The evolution of photosynthesis caused the oxygen-rich atmosphere in which we thrive today. Although the reaction centers involved in oxygenic photosynthesis probably evolved from a protein like the reaction centers

The evolution of photosynthesis caused the oxygen-rich atmosphere in which we thrive today. Although the reaction centers involved in oxygenic photosynthesis probably evolved from a protein like the reaction centers in modern anoxygenic photosynthesis, modern anoxygenic reaction centers are poorly understood. One such anaerobic reaction center is found in Heliobacterium modesticaldum. Here, the photosynthetic properties of H. modesticaldum are investigated, especially as they pertain to its unique photochemical reaction center.

The first part of this dissertation describes the optimization of the previously established protocol for the H. modesticaldum reaction center isolation. Subsequently, electron transfer is characterized by ultrafast spectroscopy; the primary electron acceptor, a chlorophyll a derivative, is reduced in ~25 ps, and forward electron transfer occurs directly to a 4Fe-4S cluster in ~650 ps without the requirement for a quinone intermediate. A 2.2-angstrom resolution X-ray crystal structure of the homodimeric heliobacterial reaction center is solved, which is the first ever homodimeric reaction center structure to be solved, and is discussed as it pertains to the structure-function relationship in energy and electron transfer. The structure has a transmembrane helix arrangement similar to that of Photosystem I, but differences in antenna and electron transfer cofactor positions explain variations in biophysical comparisons. The structure is then compared with other reaction centers to infer evolutionary hypotheses suggesting that the ancestor to all modern reaction centers could reduce mobile quinones, and that Photosystem I added lower energy cofactors to its electron transfer chain to avoid the formation of singlet oxygen.

In the second part of this dissertation, hydrogen production rates of H. modesticaldum are quantified in multiple conditions. Hydrogen production only occurs in cells grown without ammonia, and is further increased by removal of N2. These results are used to propose a scheme that summarizes the hydrogen-production metabolism of H. modesticaldum, in which electrons from pyruvate oxidation are shuttled through an electron transport pathway including the reaction center, ultimately reducing nitrogenase. In conjunction, electron microscopy images of H. modesticaldum are shown, which confirm that extended membrane systems are not exhibited by heliobacteria.

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Date Created
  • 2017

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Study of protein production, folding, crystallization and structure: survival of motor neuron protein and Fenna-Matthews-Olson protein

Description

Protein crystallization has become an extremely important tool in biochemistry since the first structure of the protein Myoglobin was solved in 1958. Survival of motor neuron protein has proved to

Protein crystallization has become an extremely important tool in biochemistry since the first structure of the protein Myoglobin was solved in 1958. Survival of motor neuron protein has proved to be an elusive target in regards to producing crystals of sufficient quality for X-ray diffraction. One form of Survival of motor neuron protein has been found to be a cause of the disease Spinal Muscular Atrophy that currently affects 1 in 6000 live births. The production, purification and crystallization of Survival of motor neuron protein are detailed. The Fenna-Matthews-Olson (FMO) protein from Pelodictyon phaeum is responsible for the transfer of energy from the chlorosome complex to the reaction center of the bacteria. The three-dimensional structure of the protein has been solved to a resolution of 2.0Å with the Rwork and Rfree values being 16.6% and 19.9% respectively. This new structure is compared to the FMO protein structures of Prosthecocholoris aestuarii 2K and Chlorobium tepidum. The early structures of FMO contained seven bacteriochlorophyll-a (BChl) molecules but the recent discovery that there is an eighth BChl molecule in Ptc. aestuarii 2K and Cbl. tepidum and now in Pld. phaeum requires that the energy transfer mechanism be reexamined. Simulated spectra are fitted to the experimental optical spectra to determine how the BChl molecules transfer energy through the protein. The inclusion of the eighth BChl molecule within these simulations may have an impact on how energy transfer through FMO can be described. In conclusion, a reliable method of purifying and crystallizing the SMNWT protein is detailed, the placement of the 8th BChl-a within the electron density and the implications on energy transfer within the FMO protein when the 8th BChl-a is included from the green sulfur bacteria Pld. phaeum is discussed.

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Date Created
  • 2010

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Characterizing the influence of amino acids on the oxidation/reduction properties of transition metals

Description

The utilization of solar energy requires an efficient means of its storage as fuel. In bio-inspired artificial photosynthesis, light energy can be used to drive water oxidation, but catalysts that

The utilization of solar energy requires an efficient means of its storage as fuel. In bio-inspired artificial photosynthesis, light energy can be used to drive water oxidation, but catalysts that produce molecular oxygen from water are required. This dissertation demonstrates a novel complex utilizing earth-abundant Ni in combination with glycine as an efficient catalyst with a modest overpotential of 0.475 ± 0.005 V for a current density of 1 mA/cm2 at pH 11. The production of molecular oxygen at a high potential was verified by measurement of the change in oxygen concentration, yielding a Faradaic efficiency of 60 ± 5%. This Ni species can achieve a current density of 4 mA/cm2 that persists for at least 10 hours. Based upon the observed pH dependence of the current amplitude and oxidation/reduction peaks, the catalysis is an electron-proton coupled process. In addition, to investigate the binding of divalent metals to proteins, four peptides were designed and synthesized with carboxylate and histidine ligands. The binding of the metals was characterized by monitoring the metal-induced changes in circular dichroism spectra. Cyclic voltammetry demonstrated that bound copper underwent a Cu(I)/Cu(II) oxidation/reduction change at a potential of approximately 0.32 V in a quasi-reversible process. The relative binding affinity of Mn(II), Fe(II), Co(II), Ni(II) and Cu(II) to the peptides is correlated with the stability constants of the Irving-Williams series for divalent metal ions. A potential application of these complexes of transition metals with amino acids or peptides is in the development of artificial photosynthetic cells.

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Date Created
  • 2014