Browsing by Author "Cooper, David"

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    Conformational Dynamics of the Glutamate Receptors via single Molecule Förster Resonance Energy Transfer
    (2015-04-21) Cooper, David; Landes, Christy F; Clementi, Cecilia; Phillips, George
    Glutamate receptors perform a critical role in the nervous system, mediating synaptic transmission through cellular membranes. These proteins have developed a quick response to agonist presence and, when activated, undergo a conformational shift that allows calcium ion transport across the membrane. In a number of neurological diseases, genetic variants can cause mutations that disrupt the balance between fast binding response and desensitization resulting in over- or under- signaling in affected cells. Herein it describes the use of single molecule Förster resonance energy transfer to observe the bound form of the glutamate receptors and subsequent dynamics. For a complete understanding of the dynamics involved in the conformational landscape, the response of the receptor to a variety of known full and partial agonists is tested. Additionally multiple receptor types are investigated to determine if the conformational pathway is conserved between the different types of glutamate receptors. The results from this project allows for a more complete understanding of the relationship between conformation and functionality of glutamate receptors.
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    Extending single molecule fluorescence observation time by amplitude-modulated excitation
    (IOP Publishing, 2013) Kisley, Lydia; Chang, Wei-Shun; Cooper, David; Mansur, Andrea P.; Landes, Christy F.
    We present a hardware-based method that can improve single molecule fluorophore observation time by up to 1500% and super-localization by 47% for the experimental conditions used. The excitation was modulated using an acousto-optic modulator (AOM) synchronized to the data acquisition and inherent data conversion time of the detector. The observation time and precision in super-localization of four commonly used fluorophores were compared under modulated and traditional continuous excitation, including direct total internal reflectance excitation of Alexa 555 and Cy3, non-radiative Förster resonance energy transfer (FRET) excited Cy5, and direct epi-fluorescence wide field excitation of Rhodamine 6G. The proposed amplitude-modulated excitation does not perturb the chemical makeup of the system or sacrifice signal and is compatible with multiple types of fluorophores. Amplitude-modulated excitation has practical applications for any fluorescent study utilizing an instrumental setup with time-delayed detectors.
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    Fast Step Transition and State Identification (STaSI) for Discrete Single-Molecule Data Analysis
    (American Chemical Society, 2014) Shuang, Bo; Cooper, David; Taylor, J. Nick; Kisley, Lydia; Chen, Jixin; Wang, Wenxiao; Li, Chun Biu; Komatsuzaki, Tamiki; Landes, Christy F.; Rice Quantum Institute
    We introduce a step transition and state identification (STaSI) method for piecewise constant single-molecule data with a newly derived minimum description length equation as the objective function. We detect the step transitions using the Student’s t test and group the segments into states by hierarchical clustering. The optimum number of states is determined based on the minimum description length equation. This method provides comprehensive, objective analysis of multiple traces requiring few user inputs about the underlying physical models and is faster and more precise in determining the number of states than established and cutting-edge methods for single-molecule data analysis. Perhaps most importantly, the method does not require either time-tagged photon counting or photon counting in general and thus can be applied to a broad range of experimental setups and analytes.
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    Photobleaching Lifetimes of Cyanine Fluorophores Used for Single Molecule Förster Resonance Energy Transfer in the Presence of Various Photoprotection Systems
    (John Wiley & Sons, Inc., 2013) Cooper, David; Uhm, Heui; Tauzin, Lawrence J.; Poddar, Nitesh; Landes, Christy F.
    We investigated various photoprotection system combinations to find the combination that optimally extended the photobleach lifetime of a Cy3/Cy5 smFRET pair attached to a DNA hairpin in a single-molecule environment. We found that the glucose/glucose oxygen-scavenging solution in combination with redox-based photostabilization solutions yielded the longest average photobleaching lifetimes
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    Single-Molecule FRET Studies of HIV TAR–DNA Hairpin Unfolding Dynamics
    (American Chemical Society, 2014) Chen, Jixin; Poddar, Nitesh K.; Tauzin, Lawrence J.; Cooper, David; Kolomeisky, Anatoly B.; Landes, Christy F.
    We directly measure the dynamics of the HIV trans-activation response (TAR)-DNA hairpin with multiple loops using single-molecule Förster resonance energy transfer (smFRET) methods. Multiple FRET states are identified that correspond to intermediate melting states of the hairpin. The stability of each intermediate state is calculated from the smFRET data. The results indicate that hairpin unfolding obeys a "fraying and peeling" mechanism, and evidence for the collapse of the ends of the hairpin during folding is observed. These results suggest a possible biological function for hairpin loops serving as additional fraying centers to increase unfolding rates in otherwise stable systems. The experimental and analytical approaches developed in this article provide useful tools for studying the mechanism of multistate DNA hairpin dynamics and of other general systems with multiple parallel pathways of chemical reactions.