WorldCat Identities

Boxer, Steven G. (Steven George) 1947-

Works: 35 works in 35 publications in 1 language and 43 library holdings
Roles: Thesis advisor, Author
Publication Timeline
Most widely held works about Steven G Boxer
Most widely held works by Steven G Boxer
Small changes big impact, ligand influence on dioxygen, semiquinone, and phenoxyl radical copper complexes by Prateek Verma( )

1 edition published in 2012 in English and held by 2 WorldCat member libraries worldwide

Reductive activation of dioxygen by copper to generate potent oxidants for multi- electron organic transformations is exploited extensively in biological systems. This thesis focuses on two types of multi-electron oxidants: Cu2O2 complexes and copper complexes with redox-active ligands. The goal of this work is to identify the influence of nitrogen containing ancillary ligands on properties of dioxygen, semiquinone and phenoxyl radical complexes of copper. This work is aimed primarily at synthetic chemists interested in rational design of ligands for creating bio-inspired oxidants and oxidation catalysts. Chapter 1 of this thesis reports the identification of a Density Functional Theory (DFT) protocol for deriving structure-property relationships in Cu2O2 complexes. Chapter 2 of this thesis applies towards modeling electronic spectra, the DFT protocols that were validated in Chapter 1 for modeling thermodynamics. Chapter 3 of this thesis describes the properties and reactivity of Cu2O2 complex generated from a new hybrid permethylated-amine-guanidine ligand based on a 1,3- propanediamine backbone (2L). Chapter 4 of this thesis describes the characterization of an intermediate (C) that is observed in both phenol hydroxylation and catechol oxidation with the SP core supported by N1, N2-di-t-butylethane-1,2-diamine (DBED). Chapter 5 of this thesis describes the influence of sulfanyl substituents on the optical and redox properties of copper-bonded phenoxyls
Developing novel therapies for gram-negative bacterial infections and glioblastoma multiforme by Tracy Curtis Holmes( )

1 edition published in 2012 in English and held by 2 WorldCat member libraries worldwide

Developing novel therapies for gram-negative bacterial infections and glioblastoma multiforme I. cloning and characterization of the guadinomine biosynthetic gene cluster II. developing a novel chemo-sensitizing agent to treat glioblastoma. This thesis explores the development of novel therapies for the treatment of two complicated problems: Gram-negative bacterial infections and glioblastoma multiforme, the most aggressive form of brain cancer. Part I of the thesis summarizes the current body of knowledge regarding guadinomines, their biosynthesis and implications for developing novel anti-infective agents. Part II of the thesis summarizes the development of the small molecule, ERW1227B, and its ability to sensitize glioblastoma cells to standard therapies. Part I. Guadinomines are a recently discovered family of anti-infective compounds produced by Streptomyces sp. K01-0509. They are the first microbial metabolites shown to inhibit the Type III Secretion System (TTSS) of Gram-negative bacteria. The TTSS is required for the virulence of many pathogenic Gram-negative bacteria including Escherichia coli, Salmonella spp., Yersinia spp., Chlamydia spp., Vibrio spp., and Pseudomonas spp. Inhibition of the TTSS can mitigate virulence which is important considering that Gram-negative bacteria infect millions each year, leading to considerable morbidity and mortality. The guadinomine (gdn) biosynthetic gene cluster has been sequenced, and encodes a chimeric multimodular polyketide synthase -- nonribosomal peptide synthetase spanning 26 open reading frames and 51.2 kb. It also encodes enzymes responsible for the biosynthesis of the unusual aminomalonyl-ACP extender unit and the signature carbamoylated cyclic guanidine. Its identity was established by genetic inactivation of the cluster, as well as heterologous expression and analysis of enzymes in the biosynthetic pathway. Identifying the guadinomine gene cluster provides critical insight into the biosynthesis of these biologically important compounds. Part II. Glioblastomas display variable phenotypes that include increased drug-resistance associated with enhanced migratory and anti-apoptotic characteristics. These shared characteristics contribute to failure of clinical treatment regimens. Identification of novel compounds that both promote cell death and impair cellular motility is a logical strategy to develop more effective clinical protocols. Previously, we described the ability of the small molecule, KCC009, a tissue transglutaminase inhibitor, to sensitize glioblastoma cells to chemotherapy. In the current study, we synthesized a series of related compounds that show variable ability to promote cell death and impair motility in glioblastomas, irrespective of their ability to inhibit TG2. Each compound has a 3-bromo-4,5-dihydroisoxazole component that presumably reacts with a nucleophilic cysteine thiol residue in one (or more) target protein(s) that have affinity for the small molecule. Our studies focused on the effects of the compound, ERW1227B. Treatment of glioblastoma cells with ERW1227B was associated with both down-regulation of the PI-3 kinase/Akt pathway, which enhanced cell death; as well as disruption of focal adhesions and intracellular actin fibers, which impaired cellular mobility. Bioassays as well as time-lapse photography of glioblastoma cells treated with ERW1227B showed cell death and rapid loss of cellular motility. Mice studies with in vivo glioblastoma models demonstrated the ability of ERW1227B to sensitize tumor cells to cell death after treatment with either chemotherapy or radiation. The above findings identify ERW1227B as a potential novel therapeutic agent in the treatment of glioblastomas
Quantitative measurement of electrostatic fields in proteins using vibrational probes by Aaron Thomas Fafarman( )

1 edition published in 2010 in English and held by 2 WorldCat member libraries worldwide

Electrostatic fields in the interior of proteins, the consequence of the charged, polar and polarizable matter they are comprised of, have been hypothesized to vary on the order of tens of megavolts per centimeter and thus to be of tremendous consequence to biological processes. It is intuitively apparent that the rate of electron transfer in photosynthesis, the rate constant for catalysis by an enzyme, the flux through an ion channel, or the affinity between a drug molecule and its target, each involving a translocation of charged or polar species, would depend strongly on the energetic contribution from the electrostatic fields exerted by the surroundings. Despite a proliferation of calculations aimed at rationalizing the energetics of these processes, there remains a paucity of direct measurements of the electrostatic fields on which these calculations depend. By Stark spectroscopy, the directional and linear sensitivity of certain vibrational transitions to externally applied electric fields has been demonstrated, and a calibration obtained, in the form of the linear Stark tuning rate. The hypothesis has been previously submitted that for such probes, incorporated into proteins, spectroscopically observed band shifts could be quantitatively translated into changes in the electrostatic fields experienced by the probe. Carbon-fluorine and carbon-deuterium oscillators are examined as probes of electrostatic field and the means to circumvent the limitations of spectral congestion for the former and low oscillator strength for the latter are demonstrated. As an alternative solution to both problems, a straightforward and general method for the incorporation of thiocyanate electric field probes at any location in a protein by post-translational cysteine modification is presented. Incorporating nitrile probes into many locations in the proteins ribonuclease S and ketosteroid isomerase, the Stark model for vibrational band shifts is evaluated more critically than has been done previously for these probes. In ribonuclease, vibrational Stark spectra are used to calibrate multiple types of nitrile-modified proteins. The results provide evidence that the simple response to external electric fields of small, nitrile-containing molecules immobilized in frozen organic glasses can be generalized to nitriles in the interior of a protein, a requisite condition for the simple interpretation of band shifts in terms of changes in the internal electrostatic field. With this point established, the accuracy of the electrostatic force model incorporated in a molecular dynamics force field is evaluated by comparing observed spectral shifts to those calculated using simulated electrostatic fields in conjunction with the Stark model. Qualitative agreement is observed. However, the simplicity of the Stark model is complicated by the possibility of direct hydrogen-bond formation to the nitrile. This limitation is overcome using a method to both detect cases where this occurs, and to quantitatively account for this effect: a comparison of nitrile chemical shifts by NMR and frequencies by IR, each calibrated in turn by a solvatochromic model. With this additional observable, we are able to confidently ascribe spectral shifts due to mutation, pH titration and ligand binding to changes in the electrostatic fields experienced by the probes. Efforts towards employing nitrile probes to measure electric fields in the complex environment of the photosynthetic reaction center are presented
DNA-mediated fusion of lipid vesicles by Bettina Van Lengerich( )

1 edition published in 2012 in English and held by 2 WorldCat member libraries worldwide

Vesicle fusion is a central process in transport and communication in biology. In neuronal transmission, synaptic vesicles carrying neurotransmitters dock and fuse to the plasma membrane of the neuron, a process mediated by a combination of several membrane anchored and soluble proteins. Fusion results in the merger of the two apposing lipid bilayers, leading to the exchange of both the lipidic and aqueous components. The fusion reaction is thought to proceed through several stages: first, the membranes are brought into close proximity (docking), second, the outer leaflets mix, but the inner leaflets and contents remain separate (hemi-fusion), and finally, the inner leaflet and contents exchange (full fusion). Due to the complex nature of the fusion reaction and the multitude of proteins involved, the mechanism of the fusion reaction is not well understood. Simplified model systems for vesicle fusion can bring insight into the mechanism by studying the fusion reaction in a more defined and controllable system. This thesis describes a DNA-based model for the protein fusion machinery. Previously, DNA-lipids were used to tether lipid vesicles to glass-supported lipid bilayers. These vesicles could be observed by fluorescence microscopy, and are laterally mobile along the plane parallel to the supported bilayer. DNA-mediated docking between vesicles was characterized, but fusion was not observed due to the fact that the DNA partners were both coupled at the 5' end, so antiparallel hybridization holds the membranes apart. In this work, a new synthesis of DNA-lipid conjugates is described which allows coupling at both the 3' and 5' end of the DNA. Incorporation of complementary DNA-lipids coupled at opposite ends mediates fusion between lipid vesicles. Vesicle fusion was measured in bulk fluorescence assays (Chapter 2 and 3), by both lipid mixing and content mixing assays. The rate of vesicle fusion showed a strong dependence on the number of DNA per vesicle, as well as the sequence of the DNA. Consistent with previous results measured for the docking reaction, fusion was faster for a repeating DNA sequence than for a non-repeating sequence that required full overlap of the strands for hybridization. The role of membrane proximity on the rate of vesicle fusion was investigated in Chapter 3 by insertion of a short spacer sequence at the membrane-proximal end of fusion sequences. The length of the spacer sequence was varied between two and 24 bases, corresponding to length scales of approximately 1-12 nm. Fusion, as measured in bulk assays by lipid and content mixing, decreases systematically as the membranes are held progressively further apart, demonstrating a clear dependence of the rate of the fusion reaction on membrane proximity. While the bulk vesicle fusion assays showed that DNA-lipids can mediate vesicle fusion, these ensemble measurements convolve the multiple steps (docking, hemifusion, and full fusion) of the fusion reaction, complicating any kinetic analysis. In order to image individual vesicle fusion events between tethered vesicles, a new tethering strategy was developed (Chapter 4). This strategy exploits the dependence of DNA hybridization on salt by covalently attaching lipid vesicles to a glass-supported lipid bilayer, then triggering DNA-mediated docking and fusion by spiking the salt concentration. The kinetics of individual vesicle fusion events were subsequently measured using a FRET-based lipid mixing assay for many vesicles (Chapter 6). An analysis of the distribution of waiting times from docking to fusion indicated that this transition occurs in a single step. A second model membrane architecture was used to study individual fusion events between vesicles and a planar bilayer (Chapters 5 and 6). This architecture uses a DNA-tethered planar free-standing bilayer as the target membrane. The kinetics of individual vesicle fusion events to this membrane patch were also consistent with a single step process, as for vesicle to vesicle fusion. In this system, it was also possible to observe content transfer of vesicles containing a self-quenched aqueous dye (Chapter 5). By analyzing the diffusion profile of the dye, it was shown that the dye indeed is transferred into the region below the planar membrane patch, and is not released into the solution above the patch due to vesicle rupture or leakage
Single-molecule fluorescence and super-resolution imaging of Huntington's disease protein aggregates by Whitney Clara Duim( )

1 edition published in 2012 in English and held by 2 WorldCat member libraries worldwide

Single-molecule, super-resolution fluorescence microscopy is a powerful technique for studying biological systems because it reveals details beyond the optical diffraction limit (on the 20-100 nm scale) such as structural and conformational heterogeneity. Further, single-molecule imaging measures distributions of behaviors directly through the interrogation of many individual molecules and reports on the nanoscale environment of molecules. Sub-diffraction imaging adds increased resolution to the advantages of fluorescence imaging over the techniques of atomic force microscopy and electron microscopy for studying biological structures, which include imaging of large fields of view in aqueous environments, specific identification of protein(s) of interest by fluorescent labeling, low perturbation of the system, and the ability to image living systems in near real-time (limited by the time required for super-resolution sequential imaging). This dissertation describes the application of single-molecule and super-resolution fluorescence imaging to studying the huntingtin (Htt) protein aggregates that are a hallmark of Huntington's disease and that have been implicated in the pathogenesis of the disease. The intricate nanostructures formed by fibrillar Htt aggregates in vitro and the sub-diffraction widths of individual fibers mark the amyloids as important targets for high-resolution optical imaging. The characterization of Htt aggregate species is critical for understanding the mechanism of Huntington's disease and identifying potential therapies. Following an introduction to single-molecule, super-resolution imaging and Huntington's disease in Chapter 1, Chapter 2 describes the single-molecule methods, experimental techniques, Htt protein sample preparations, and data analysis performed in this dissertation. Chapter 3 discusses the development of super-resolution imaging of Htt protein aggregates and the validation of the images by atomic force microscopy. Chapter 4 continues the study of Htt by one- and two-color super-resolution with imaging of Htt protein aggregates over time from the initial protein monomers to the large aggregate assemblies of amyloid fibers. In Chapter 5, I detail our progress to-date in studying the earliest stages of Htt aggregation using zero-mode waveguide technology. Chapter 6 concludes the dissertation with a discussion of the results from additional projects comprising the effect of chaperonin proteins on Htt aggregation, extension of super-resolution Htt imaging to three dimensions, and cellular imaging of Htt aggregates. The future directions for these exciting projects are summarized with the expectation that research efforts directed in these areas will contribute to our understanding of Htt aggregation and Huntington's disease
Nanopillar structures for cellular interface by Chong Xie( )

1 edition published in 2011 in English and held by 2 WorldCat member libraries worldwide

Abstract The small scale of nano-materials makes them one of the best man-made candidates to interact with biological systems at subcellular or even molecular level. It has been the focal point of the research interests to interfacing live cells with one dimensional nanostructures, such as nanowires and nanopillars. In my Phd research, I have utilized nanopillar based structures and devices to interface biological cells electrically, optically and mechanically. 1. We achieve improved electric interface between biological cells and solid state device by using arrays of vertically aligned nanopillar electrodes. Their tight attachment to the cell membrane allows us to acquire intracellular-like action potential signals non-destructively from cultured cardiomyocytes, which is responsible for various important cellular functions. 2. We demonstrate below-the-diffraction-limit observation volume in vitro and inside live cells by using vertically aligned silicon dioxide nanopillars. With a diameter much smaller than the wavelength of visible light, a transparent silicon dioxide nanopillar embedded in a nontransparent substrate restricts the propagation of light and affords evanescence wave excitation along its vertical surface. This effect creates highly-confined illumination volume that selectively excites fluorescence molecules in the vicinity of the nanopillar. We show that this nanopillar illumination can be used for in vitro single molecule detection with high fluorescence background. In addition, we demonstrate that vertical nanopillars interface tightly with live cells and function as highly localized light sources inside the cell. Furthermore, chemical modification of the nanopillar surface provides a unique way to locally recruit proteins of interest and simultaneously observe their behavior within the complex, crowded environment of the cell. 3. We engineer and fabricate vertically nanopillar arrays, and culture various types of cells atop. We study the cell growth pattern in presence of nanopillar arrays, including attachment, migration, etc. We also design patterned nanopillar arrays and utilized them to guide and control cell growth via cell-nanopillar interaction
Developing novel lipid architectures as a platform for membrane biophysics by Minsub Chung( )

1 edition published in 2011 in English and held by 2 WorldCat member libraries worldwide

Many cellular processes including cell-cell communications and regulated membrane transport are mediated by membrane proteins and depend upon the ability of lipid membranes to be a differentially permeable barrier. However, the roles and function of membrane proteins are often difficult to study due to the complexity of the native membranes and lack of reliable and flexible artificial model lipid membranes. Supported lipid bilayers (SLB) have been used as a model system to study biological membrane behavior and the structure and function of membrane proteins and receptors in a simpler context apart from the complex cellular environment. Although SLBs have the advantages of simple formation, easy handling and are well-suited for investigation by a suite of surface sensitive methods due to their planar geometry, the close proximity of the lower leaflet to the solid support often leads to unfavorable interactions with integral membrane proteins. This causes distortion of the protein conformation and possible loss of its reactivity and function. Moreover, this interaction with the substrate often traps proteins and reduces their mobility in the membranes. Recognizing this limitation, we have developed a new model membrane architecture in which the DNA-tethered lipid bilayer is either to fixed DNA on a surface or to laterally mobile DNA displayed on a supported bilayer. This separates the lipid membranes from surface interactions and provides a more favorable environment for integral membrane protein with large globular domains. With mobile DNA hybrid tethers, stable tethered bilayers were made with specific lipid composition, while those with fixed tethers are stable regardless of membrane composition. The mobile tethers between a tethered and a supported lipid bilayer offer a particularly interesting architecture for studying the dynamics of membrane-membrane interactions. By careful choice of composition, improved stability was obtained and we can investigate the lateral segregation of DNA hybrids when different lengths are present. Based on a theoretical model, the effects of population, length and affinity of DNA complexes are simulated and described. This model system captures some of the essential physics of synapse formation and is a step toward understanding lipid membrane behavior in a cell-to-cell junction. To demonstrate the excellent environment provided by DNA-tethered membranes for studying transmembrane proteins free from any surface interactions, the behavior of a transmembrane protein, the photosynthetic reaction center, reconstituted in the DNA-tethered membranes is investigated. Inspired by DNA-mediated membrane fusion studies of our group, we applied the DNA-machinery to achieve fusion of small (~ 100 nm) proteoliposomes for delivery of membrane proteins to either giant vesicles or DNA-tethered planar lipid membrane patches. The diffusion behavior of delivered proteins is measured and compared with those in supported bilayers. Also, the protein activity and orientation before and after fusion is analyzed. This will offer a feasible method to incorporate intact membrane proteins to already formed model membranes. In addition, the behavior of proteins during the fusion event will provide insight into the mechanism of DNA-mediated lipid membrane fusion. The geometry of our model membrane system directly mimics that of a neuronal synapse. We expect that this architecture will be readily transferable to other model membrane fusion systems, including systems using reconstituted SNARE proteins. Consequently, it will be of considerable interest to a wide range of researchers
The development of techniques for three-dimensional super-resolution fluorescence microscopy and their application to biological systems by Michael Anthony Thompson( )

1 edition published in 2011 in English and held by 2 WorldCat member libraries worldwide

Fluorescence microscopy is one of the most widely used tools in cell biology due its intrinsically high detection sensitivity coupled with the ability to genetically label proteins and other cellular structures with fluorescent tags. However, the resolution of fluorescence microscopy has historically been limited to about 200 nm laterally and 800 nm axially because of the diffraction limit of visible light. In the past five years, imaging below the diffraction limit ("super-resolution imaging") by localizing single fluorophores, one at a time (1-3), has opened a wide a variety of new biological systems for study. This Dissertation is a collection of both techniques for two and three dimensional super-resolution imaging as well as applications in bacterial and yeast imaging. References 1. Betzig E, et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313: 1642-1645. 2. Hess ST, Girirajan TPK & Mason MD (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91: 4258-4272. 3. Rust MJ, Bates M & Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3: 793-795
Atomistic insights into microbial biofilms by Courtney Reichhardt( )

1 edition published in 2016 in English and held by 1 WorldCat member library worldwide

Most microbes live as multi-cellular communities termed biofilms. This lifestyle protects microbes against harsh conditions including antibiotic treatment and host immune responses. Within biofilms, microbial cells are entangled in a self-secreted extracellular matrix (ECM) that is rich in biopolymers such as fibrillar proteins and polysaccharides. This extracellular material is key to the characteristic properties of biofilms. Despite the prevalent roles that biofilms play in infections, a molecular-level understanding of the insoluble matrix components or the interactions between the ECM components has yet to be described. Biofilms and ECM are neither soluble nor crystalline, which poses challenges to analysis by traditional biochemical techniques. Solid-state nuclear magnetic resonance (NMR) is uniquely suited to study such complex systems because it provides quantitative information about chemical composition and also the spatial relationships of the components without requiring degradative sample preparation. Using solid-state NMR, we previously elucidated that the insoluble ECM produced by a uropathogenic strain of Escherichia coli called UTI89 is composed of two biopolymers: a functional amyloid called curli and modified cellulose. The purpose of this study is to elucidate quantitative information about microbial biofilm composition and structural information about biofilm constituents. Additionally, we aim to achieve an understanding of how the chemical and biophysical properties of specific ECM components contribute to the overall ECM architecture. To this end, we pursued three intersecting avenues with a primary focus on the bacterial strain UTI89, although we also determined quantitative parameters of additional microbial biofilms. In the first approach, we explored the dye binding properties of the biofilm constituent and functional amyloid called curli. The ability to specifically stain ECM components has been a key step in traditional investigations of biofilms, and we sought to provide a foundation to similarly study curli. In the second approach, we developed a means to spectroscopically annotate chemically complex ECM composition of the important human pathogens Vibrio cholerae and Aspergillus fumigatus using solid-state NMR. Finally, we provided novel biophysical and structural details of specific ECM components to better understand how these biopolymers interact to form robust ECM networks. Looking forward, we have begun to utilize solid-state NMR to provide a global accounting of the architecture of the UTI89 ECM. Together these studies have provided important quantitative parameters of biofilm composition and structural information of ECM components. Our analysis has wide-ranging implications for understanding the fundamental mechanisms of biofilm formation and for the development of functional biopolymeric materials
Three-dimensional single-molecule microscopy of bacterial regulatory proteins within a pole-localized microdomain by Alex von Diezmann( )

1 edition published in 2018 in English and held by 1 WorldCat member library worldwide

Since the first optical detection of a single molecule 29 years ago, the development of single-molecule microscopy and spectroscopy has revolutionized the study of complex chemical systems. As reviewed in Chapter 1, by imaging and computationally localizing individual fluorescent dyes or proteins within a sample, their positions can be localized with typical precisions (10-40 nm) an order of magnitude or better than the optical diffraction limit of visible light (~250 nm laterally and ~500 nm axially). This technique is critical to super-resolution fluorescence microscopy and single-molecule tracking, which are now regularly used to measure the nanoscale structures, biomolecular motions, and stochastic chemical processes underlying the biology of cells. This dissertation comprises two intertwined single-molecule imaging projects: 1) optical and analytical methods development for three-dimensional (3D) single-molecule tracking and super-resolution microscopy, and 2) the application of these methods to understand the nanoscale organization and dynamics of proteins at the poles of the bacterium Caulobacter crescentus. Without modification, a single-molecule microscope only improves imaging resolution in the lateral (xy) dimension, but biological cells are intrinsically 3D. To improve the imaging resolution in z, the detection path of a standard widefield microscope can be modified using Fourier processing to encode z position in the pattern of light formed by each fluorescent emitter and detected on the camera. Chapter 2 reviews the development of a two-color 3D single-molecule microscope that uses the double-helix point spread function pattern to encode 3D position, while Chapter 3 describes how to correctly align and to calibrate the fine aberrations of such a microscope to achieve nanoscale imaging accuracy in multiple color channels simultaneously. The bacterium Caulobacter crescentus is a model organism for the study of cell polarization and asymmetric cell division. Chapters 4 and 5 describe work performed in collaboration with Prof. Lucy Shapiro and her laboratory in the Department of Developmental Biology in the Stanford University School of Medicine to study how the tips, or poles, of Caulobacter cells use proteins to act as nanoscale spatial landmarks that polarize cells and induce spatially organized development. The polar organizing protein PopZ is one such critical landmark, and Chapter 4 describes results obtained from 3D super-resolution imaging of PopZ. Such imaging showed that PopZ forms 150-200 nm space-filling polar microdomains of roughly uniform density, and that proteins of the chromosome partitioning machinery (ParA and ParB) exhibit different spatial behaviors (recruitment vs. tethering) relative to the PopZ microdomain depending on their biochemistry and role in the chromosome replication process. Chapter 5 discusses the combination of single-molecule tracking and super-resolution imaging to study the polar localization of the signaling molecules of that activate the master regulator protein CtrA. Precise 3D imaging and tracking showed that PopZ acts as a selectively permeable localization hub that slows the motion of signaling proteins. In combination with reaction-diffusion modeling and transcriptional assays, these microscopic measurements indicated that the PopZ microdomain acts to sequester the CtrA signaling pathway within the pole and spatially pattern transcriptional output within the predivisional Caulobacter cell
Trafficking and axodendritic transcytosis of BDNF in hippocampal neurons by Wenjun Xie( )

1 edition published in 2014 in English and held by 1 WorldCat member library worldwide

Brain derived neurotrophic factor (BDNF) plays a key role in the growth, development and maintenance of the central and peripheral nervous systems. Exogenous BDNF activates its membrane receptors at the axon terminal, and subsequently sends regulation signals to the cell body. To understand how BDNF signal propagates in neurons, it is important to follow the trafficking of BDNF after it is internalized at the axon terminal. Here we labeled BDNF with bright, photostable quantum dot (QD-BDNF) and followed the axonal transport of QD-BDNF in real time in hippocampal neurons. We showed that QD-BDNF was able to bind BDNF receptors and activate downstream signaling pathways. When QD-BDNF was applied to the distal axons of hippocampal neurons, it was observed to be actively transported toward the cell body at an average speed of 1.11 ± 0.05 [mu] m/s. A closer examination revealed that QD-BDNF was transported by both discrete endosomes and multivesicular body-like structures. Our results showed that QD-BDNF could be used to track the movement of exogenous BDNF in neurons over long distances and to study the signaling organelles that contain BDNF. Surprisingly, not all QD-BDNF ended up in lysosomes in the cell body for degradation. Instead, some BDNF was transported to dendrites possibly for signaling purposes. BDNF transcytosis challenges the model that neurotrophic action is terminated by rapid degradation of the neurotrophin molecule after delivery of the trophic signal. It is postulated that BDNF as well as other neurotrophic factors might be recycled and targeted to dendrites as endogenous neurotrophins for the purpose of releasing. To develop a comprehensive understanding of the molecular mechanisms of BDNF transcytosis, it is important to follow BDNF retrograde transport in axons toward the cell body, axodendritic translocation, properties of the BDNF endosomes transcytosed into dendrites, and study whether these processes are activity dependent. Using QD for monitoring BDNF transcytosis through neurons requires that the labeling method does not perturb the biological function of BDNF. Meanwhile, to observe the trafficking of QD-BDNF internalized from the axon terminal, it is necessary to polarize the growth of axons into a fluidic isolated environment and separate from cell body and dendrites. With modifications and coating with neurite guidance molecules in the channels, the microfluidic platform can be improved to fit the purpose of observing BDNF transcytosis in a more controlled environment. By combining fluorescent labeled BDNF, microfluidic neuron culture platform and dual-color imaging, we found that (1) After BDNF internalized from axon terminal transport retrogradely into soma, most goes to degradation but some enters dendrites and ends up at different locations with its receptor TrkB; (2) Cargos transcytosed from axon terminal can be directed into dendrites and transport in similar manner as endogenous protein cargos
A report on picosecond studies of electron transfer in photosynthetic models by T. L Netzel( Book )

1 edition published in 1980 in English and held by 1 WorldCat member library worldwide

Functionally relevant solvation dynamics in the protein interior by William James Childs( )

1 edition published in 2010 in English and held by 1 WorldCat member library worldwide

Functionally relevant solvation dynamics in the protein interior Stanford University, 2010. The role of solvent in the kinetics and thermodynamics of charge transfer reactions in simple solvents is well studied. However, biologically relevant systems often involve chemical reactions not in simple aqueous environments, but within a protein interior that constitutes an organized solvent environment. The role of the protein environment during catalysis and charge transfer reactions continues to be debated. To determine the role of the protein environment during charge transfer reactions, experiments presented in this dissertation were designed to measure the time-resolved response of a protein environment to sudden electronic perturbation. Specifically, time-resolved fluorescence measurements were used to determine the solvation response of a chromophore bound in the protein interior. An unnatural amino acid containing a fluorescent side chain, aladan, was synthetically incorporated into seven sites of a small globular protein, GB1, in order to determine the degree of solvation in different regions of a single protein. In all regions, the solvation dynamics showed an ultra-fast solvation response on the subpicosecond timescale. However, in addition to the subpicosecond dynamics, buried sites also showed relatively long solvation behavior over nanoseconds. mPlum, a variant of the red-fluorescent protein DsRed, was produced by a directed evolution experiment in the Roger Tsien lab. Ultra-fast fluorescence upconversion experiments measured a solvation response in mPlum. Examining the comprehensive library of mutants generated by the directed evolution experiment identified a single hydrogen bond between glutamic acid 16 and the chromophore to be the origin of the solvation process. The possibility of localizing a solvation response to a single interaction is only possible in an organized solvent system like a protein interior. The catalytic relevance of solvation dynamics was studied with a light-driven reaction analog for isomerisation of [delta]5-3-ketosteroids in ketosteroid isomerase. The light-driven analog consists of a photoacid bound to the active site of ketosteroid isomerase. In the ground state, the photoacid electrostatically resembles the reaction intermediate. In the excited state, the photoacid resembles the starting material. The solvation response recorded from the fluorescing photoacid shows a dramatically reduced solvation capacity and slower dynamics than observed with the same fluorophore in solution. Solvation within the protein occurs on the nanosecond timescale compared to picoseconds in solution. The active site of ketosteroid isomerase does show dynamic electrostatic behavior during the simulated reaction; however, the response is among the slowest recorded. The resistance to electrostatic perturbation suggests an electrostatically preorganized environment which does not significantly reorganize during catalysis
Green fluorescent protein and its 10th [beta]-strand : controlling protein-peptide interactions with light by Keunbong Do( )

1 edition published in 2014 in English and held by 1 WorldCat member library worldwide

Truncated green fluorescent protein (GFP) that is refolded after removing the 10th [beta]-strand (s10) can readily bind to a synthetic strand to recover the absorbance and the fluorescence of the whole protein. This allows rigorous experimental determination of thermodynamic and kinetic parameters of the split system including the equilibrium and the association/dissociation rate constants, which enables residue-specific analysis of protein-peptide interactions. The equilibrium constant obtained from the ratio of the two rate constants agrees with that directly estimated from the binding isotherm, which supports the one-to-one binding model. In measuring the dissociation rate of s10, it was discovered that the strand can photodissociate, where the dissociation rate is greatly increased by light irradiation. We found that in the overall photodissociation process, a thermal step follows light-activation of the molecule, and the thermal activation barrier was determined through an Arrhenius plot of the rate constants acquired at a saturating light intensity. The thermal step could be selectively affected by mutating residue 209 on s10 and by changing the solvent viscosity, which suggests that the thermal step is the actual dissociation of s10. In the process, we found that the quantum yield of photodissociation can be enhanced by increasing the temperature or lowering the viscosity of the solvent, or by introducing mutations such as K209Q or Y203T. For the light-activation step, cis-trans isomerization of the chromophore could be suggested as the underlying mechanism referring to the previous work on truncated GFP with strand 11 removed (Kent and Boxer, 2011). Photodissociation links photoexcitation of the protein to its conformational change, which opens the possibility of using GFP in caging. Finally, GFP variants carrying one extra s10 were created and characterized, and their possible applications were explored. These proteins can fold with either one or the other s10, and the ratio of the two folded forms, unambiguously distinguished by their resulting colors, can be systematically modulated by mutating the residues on s10 or by changing the lengths of the two inserted linker sequences that connect each s10 to the rest of the protein. Exploiting studies on photodissociation, ratiometric protease sensors were designed from the construct by engineering a specific protease cleavage site into one of the inserted loops, where the bound s10 is replaced by the other strand upon protease cleavage and irradiation with light to switch its color. Since the conversion involves a large spectral shift, these genetically encoded sensors display very high ratiometric dynamic range. Further engineering of this class of proteins guided by mechanistic understanding of the light-driven process will enable interesting and useful applications of the protein
Studies of electrostatics and hydrogen bonds in human aldose and aldehyde reductase using a nitrile-containing inhibitor by Lin Xu( )

1 edition published in 2013 in English and held by 1 WorldCat member library worldwide

Human aldose reductase (hALR2) and human aldehyde reductase (hALR1) are both aldo-keto reductases with highly conserved tertiary structures. hALR2 is involved in the development of long-term, secondary diabetes complications. In light of the biomedical interest in developing selective hALR2 inhibitors against hALR1, nitrile vibrational probes were introduced into hALR2 and hALR1 through the binding of nitrile-containing inhibitors. These probes can provide information on microenvironments (e.g. electrostatics, hydrogen bonds) in the specificity pockets of similar proteins and help us understand inhibitor selectivity mechanisms. A new nitrile-containing inhibitor was designed and synthesized. The x-ray structure of its complex with wild type (WT) hALR2, along with cofactor NADP+, was determined. The nitrile is found to be in close proximity to T113, consistent with a hydrogen bond interaction. Two vibrational absorption peaks were observed at room temperature in the nitrile region when the inhibitor binds to WT hALR2, which were empirically assigned to hydrogen bonded and non-hydrogen bonded populations. IR studies on hALR2 mutants, classical molecular dynamics (MD) simulations, temperature dependent IR measurements, and 13C NMR-IR correlation experiments provide consistent, supportive evidence for this assignment from different perspectives. Hydrogen bonding interactions usually help to improve inhibitor potency; meanwhile, it also complicates the simplest analysis of vibrational frequency shifts as being due solely to electrostatic interactions through the vibrational Stark effect (VSE). VSE spectroscopy is an experimental technique which allows direct measurements of protein electrostatic fields. In order to examine the role of electrostatics as a possible selectivity determinant, VSE spectroscopy was used to measure electric fields in the active sites of hALR2 and hALR1 with nitrile vibrational probes. X-ray structures of two mutants, hALR2:T113A and hALR1:Y115A, in complex with the inhibitor and NADP+, were solved. Mutations to amino acid residues near the nitrile probe were made to convert the specificity pocket of one protein to the other, and vibrational frequency shifts were used to quantify the electrostatic differences in these two structurally similar proteins. MD simulations were performed to calculate protein electric fields, which were compared with measured fields. The applications of VSE spectroscopy in the investigation of important biological processes such as inhibitor selectivity are discussed
Two-step laser mass spectrometric investigations of terrestrial and extraterrestrial samples by Amy Louise Morrow( )

1 edition published in 2012 in English and held by 1 WorldCat member library worldwide

The studies detailed in this thesis center around the application of laser mass spectrometry to a variety of terrestrial and extraterrestrial samples and their polycyclic aromatic hydrocarbon (PAH) contents. Though PAHs are ubiquitous in space, on Earth and, indeed, in our everyday lives, determination of characteristic PAH signatures in samples of interest can provide important clues to the mechanisms of asteroid interactions, answer questions about experimental contamination, and even help to decipher the riddles of extraterrestrial life. Specifically, this thesis describes the use of two-step laser desorption laser ionization mass spectrometry (L2MS) to: (i) determine that photochemical reactions are not a source of contaminating aromatics in NASA's Stardust mission; (ii) confirm that meteorites collected in the area of a witnessed near-Earth object (NEO) impact were indeed part of the parent asteroid; (iii) contribute to the on-going question of biotic or abiotic formation of carbonate/magnetite globules in mineralogical formations; and (iv) show that L2MS is a promising alternative to laser desorption/ionization mass spectrometry (LDI-MS) for analytes prone to gas-phase aggregation
Networked to the catalytic core : structure-function studies of RNA enzymes reveal distinct connections from the periphery by Tara Lynn Benz-Moy( )

1 edition published in 2012 in English and held by 1 WorldCat member library worldwide

The past two decades of functional and structural studies on RNA enzymes have largely focused on revealing active site interactions between ribozymes and their substrates. More recently, active site interactions among ribozyme residues themselves and the substrate have revealed an intricate network of interactions in and around the active site that involves metal ions, metal ion ligands, hydrogen bonding, and stacking interactions. Does this network extend into the broader structure of the RNA? The current studies in this thesis are aimed at understanding how the overall structural scaffold of RNA is involved in catalysis. Peripheral elements that are brought into close proximity via long-range tertiary contacts surround the catalytic core of most group I introns. In the well-studied Tetrahymena group I intron, ablation of each of five long-range tertiary contacts destabilizes the folded ribozyme, indicating a role for these tertiary contacts in overall stability, as expected. But once folded, three of the five tertiary contact mutants exhibit distinct functional roles in catalysis. Structural changes distal from the mutation site, revealed by hydroxyl radical footprinting, suggest that that these contacts are coupled to the catalytic core from a distance. These structural data combined with X-ray crystal structures and phylogenetic data suggest a number of networks that structure functional sites by using long-range tertiary contacts to position rigid helices and their more local tertiary interactions relative to the rest of the RNA. Preliminary data that tests these hypotheses suggest that some, but not all, of these networks between the periphery and the functional sites may be conserved among group I introns and that long-range tertiary contacts as well as more local tertiary interactions such as base triples may act redundantly to maintain the structure of the same regions of RNA. This redundancy could allow RNAs that change conformation over their reaction cycles to rearrange only a few tertiary contacts while maintaining a global fold. With the studies described herein, we are poised to understand the structural networks that couple the peripheral regions of RNA to its catalytic sites. These insights may lead to a better understanding of how structural networks of communication are exploited to facilitate multi-step processes that are driven by more complex RNA enzymes like the ribosome and the spliceosome
When membranes collide : studying vesicle fusion using a model system based on DNA hybridization by Robert James Rawle( )

1 edition published in 2014 in English and held by 1 WorldCat member library worldwide

Membrane fusion consists of a complex rearrangement of lipids and proteins that results in the merger of two lipid bilayers. This fusion reaction is central to many biological processes, including endo- and exocytosis and the transfer of membrane proteins between cellular compartments. The process of vesicle docking and fusion is mediated by formation of the SNARE protein complex, made up of recognition partners on the vesicle and target membranes, with many other accessory proteins assisting or regulating the process. Although extensively studied, essential questions about vesicle fusion, including the number of components involved and the precise physical mechanism, are not well understood. Due to the complexity of the fusion reaction and the proteins involved, reductionist model systems can complement in vivo data to yield a better understanding of this biological process. This dissertation describes the development and study of a reductionist model system that employs synthetic DNA-lipid conjugates as surrogates for the SNARE machinery. This model system affords easy control over DNA sequence, binding geometry, and length--factors less easily probed in SNARE-mediated fusion--and it allows us to examine how fusion proceeds once the vesicle and target membranes are brought close together in the absence of accessory factors. Previous work has demonstrated that hybridization of complementary DNA-lipid pairs on different vesicles can enable fusion between small vesicles in bulk. This dissertation expands upon that previous work to develop assays to study DNA-mediated vesicle fusion on the individual event level. These assays are then used to ask mechanistic questions about the DNA-mediated fusion reaction that were not previously addressable
Excited states, electron transfer reactions and dimers of chlorophyll derivatives and related model systems by Steven G Boxer( )

1 edition published in 1976 in English and held by 1 WorldCat member library worldwide

Ultrafast time-resolved infrared spectroscopy of molecular monolayers and solute-solvent complexes by Daniel Edward Rosenfeld( )

1 edition published in 2012 in English and held by 1 WorldCat member library worldwide

Ultrafast time-resolved infrared spectroscopy has been a powerful tool in resolving and studying ultrafast motions in bulk chemical and biological systems. The utility of ultrafast time-resolved infrared spectroscopy is illustrated through two studies of solute-solvent complexes. The same experimental methods used to study bulk systems are then extended to study surface systems through the development of both surface molecular probes and new spectroscopic techniques. Ultrafast polarization and wavelength selective IR pump-probe spectroscopy is used to measure the inertial and long time orientational dynamics of pi-hydrogen bonding complexes. The complexes studied are composed of phen-d-ol (phenol-OD) and various pi-base solvents with different electron donating or withdrawing substituents. The inertial motion is found to be insensitive to the strength of the hydrogen bond, but highly sensitive to the local solvent structure as reported on by inhomogeneous line broadening. The local solvent structure therefore acts as the controlling influence in determining the extent of inertial orientational relaxation, and thus the angular potential. Variation in the pi-hydrogen bond strength is of secondary importance. Hydrogen bonded complexes between phenol and phenylacetylene are studied using ultrafast two-dimensional infrared (2D IR) chemical exchange spectroscopy. Phenylacetylene has two possible pi-hydrogen bonding acceptor sites (phenyl or acetylene) that compete for hydrogen bond donors in solution at room temperature. The chemical exchange process occurs in ~5 ps, and is assigned to direct hydrogen bond migration along the phenylacetylene molecule. The observation of direct hydrogen bond migration can have implications for macromolecular systems. 2D IR vibrational echo spectroscopy and heterodyne detected transient grating (HDTG) spectroscopy (an ultra-sensitive analog of pump-probe spectroscopy) are developed as means of study of the structural and vibrational dynamics of surfaces. The surfaces studied are silica surfaces functionalized with a transition metal carbonyl complex, tricarbonyl (1,10)-phenanthroline rhenium chloride. The functionalization process produces chromophore surface density of 1-2 × 10^14 per cm squared. The high surface density achieved indicates that energy transfer between molecules on the surface could impact the experimental observables probed in 2D IR and HDTG spectroscopy. The theory of excitation transfer induced spectral diffusion has been developed and is capable of calculating the effect of the energy transfer on any spectroscopic observable through a master equation approach. Initial estimates of surface structural dynamics, based on both experimental 2D IR data and theoretical calculations, showed sub-100ps structural dynamics in the molecular monolayers even without the presence of solvent. Furthermore, solvent is shown to accelerate the structural dynamics in a manner that is different from that of bulk solution. Additional surface density dependent experiments indicate the negligible nature of excitation transfer even in these dense systems. The functionalized molecular monolayers are found to have a ~40 ps structural dynamics relaxation time in the absence of solvent. Further investigation of the effects of solvents on the RePhen(CO)3Cl monolayers has been carried out. Immersion in solvent is found to change the infrared spectrum, structural dynamics and vibrational dynamics in ways that differ from the changes evidenced in the bulk. The monolayers were immersed in both solvents that can dissolve RePhen(CO)3Cl and those that cannot. For both hexadecane and D2O, which cannot dissolve the headgroup, the structural dynamics of the monolayer are slowed by the presence of solvent while the vibrational dynamics are not impacted. Polar organic solvents, which can dissolve the headgroup, accelerate the dynamics. Dimethylformamide (DMF) is found to have a particularly strong effect on the structural dynamics of the monolayers, accelerating the timescale from 40 ps to 15 ps, yet DMF has little impact on the vibrational dynamics. Chloroform is found to enhance the vibrational lifetime of the CO symmetric stretch of the RePhen(CO)3Cl headgroups in the monolayer by 50%. These results indicate that the properties of thin films can be modified by the presence of solvent, even in the case when the solvent is repelled by the monolayer
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