Browsing by Subject "Electron paramagnetic resonance"

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    Site-directed modifications of myosin
    (2013-06) Moen, Rebecca Jane
    Myosins are a diverse class of molecular motors responsible for movement in all eukaryotic cells. The conversion of chemical energy from ATP hydrolysis into mechanical force produces movement along an actin filament. The mechanism of movement is involved in muscle contraction as well as various cellular processes including cytokinesis, adhesion, and vesicle transport. All myosins contain three functionally important domains: the catalytic head domain (CD), the light chain or lever arm domain (LCD), and the tail. The catalytic head domain is very similar between all classes of myosins, containing the site of ATP binding and hydrolysis and the actin-binding interface. The tail domain of myosins are highly divergent, containing either coiled-coil domains, individual subdomains, or both, that confer each myosin's specific function and cellular localization. The biochemical steps of ATP hydrolysis in myosins are accompanied by a sequence of structural transitions. A large-scale conformational change within the myosin molecule occurs where the LCD, functioning as a lever arm, rotates relative to CD. In muscle myosin, this large-scale conformation change is associated with a transition of the actomyosin complex from a state of disordered, weak actin binding to a state of ordered, strong actin binding. This research focuses on two functionally important domains within the myosin molecule: the catalytic domain and the tail domain. First, the structural transitions that occur within the myosin II catalytic domain during the actomyosin ATPase cycle are investigated using a combination of biochemical and spectroscopic approaches, specifically studying how various chemical modifications (chemical crosslinking, oxidative modifications including methionine oxidation and glutathionylation) produce functional and structural changes. Chemical crosslinking is used to capture a dynamic intermediate in the myosin ATPase cycle, resembling a weak binding state, which is defective in actomyosin functional interaction and is dynamically disordered when bound to oriented actin. In vitro oxidative modification of the myosin catalytic domain, as a model for aging and oxidative stress in muscle shows chemical, functional, and structural perturbations are predominantly caused by a specific methionine residue in the actomyosin binding interface. These combined results illustrate a crucial role in proper actin binding cleft structural dynamics in myosin function. Modification of dynamics in this region, either by crosslinking or oxidation at critical residues in cleft, affect muscle function by interfering with the critical structural transitions necessary for actomyosin functional interaction. The focus then shifts to the tail domain of myosin VII, again using biochemical and spectroscopic approaches to elucidate the functional and structural properties of a myosin tail subdomain, the MyTH/FERM domain.
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    Ultra-low Frequency Electron Paramagnetic Resonance Based on Longitudinal Detection and Fictitious Field Modulation
    (2021-07) TANG, XUEYAN
    In magnetic resonance imaging, the signal intensity is directly affected by the external field strength and the spin system properties. It is challenging to acquire signal from electron spins at ultra-low field because of their short relaxation times: the weak signal would decay away before it can be captured. The longitudinal detec- tion (LOD) of electron paramagnetic resonance (EPR) offers an alternative solution. Instead of measuring the transverse magnetization, LOD measures the longitudinal magnetization along the polarizing field direction. By placing the receive coil or- thogonal to the transmit coil and tuning it to a different frequency range, an LOD EPR system can have superior resilience against transmit cross-talk and enables si- multaneous transmit and receive. As a result, the EPR signal from spins with short relaxation times can be recorded with high signal to noise ratio. This thesis presents an ultra-low field LOD EPR measurement system. A novel method of inducing signal from longitudinal magnetization is described: fictitious field modulation. When viewed in a rotating frame of reference, a transverse-plane radiofrequency (RF) field manifests as a longitudinal field component called the fic- titious field. By modulating the RF field and thus the fictitious field, detectable longitudinal magnetization patterns are measurable. Furthermore, via combining fictitious-field modulation and longitudinal detection, this work demonstrates EPR spectroscopy and one-dimensional imaging. This custom-built LOD system oper- ates at an ultra-low frequency (24 MHz) for detecting electron spins with short (∼nanoseconds) relaxation times. Simultaneous transmit and receive with low trans- mitter leakage level (∼80 dB isolation) is also achieved. Finally, a different version of this system is introduced for measuring short relaxation times at ultra-low magnetic field, the principle of which is derived from the fictitious field theory.