Browsing by Subject "EPR"

Now showing 1 - 9 of 9
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    3D Orientation of Alpha Helix in Muscle Myosin Regulatory Light Chain Using Bifunctional Electron Paramagnetic Resonance
    (2021-06) Savich, Yahor
    Muscle contraction is a coordinated work of nanometer-sized force generators, myosin molecules. These molecules are out of equilibrium: they use the energy stored in the form of ATP to move collectively along the track protein actin. The myosin molecules transfer their work via lever arms that connect force generators to their cargo. Orientation of these lever arms has been studied thoroughly since 1) their structural dynamics is fundamental for understanding the muscle contraction and 2) their particular orientations are associated with disease states of cardiac and skeletal muscle. Electron microscopy, fluorescence polarization, and X-ray diffraction have provided insight into the structure of muscle, but there is still no high-resolution data of the vertebrate lever arm orientation available at ambient (not vitrified or crystallized) conditions. The present work establishes a method of measuring the orientation of the alpha helices in three dimensions using electron paramagnetic resonance (EPR). Chapter 3 introduces the use of EPR with bifunctional spin labels attached to different helices of the myosin regulatory light chain (RLC) protein with and without ATP. Demembranated skeletal muscle fibers were aligned with the slowly-varying magnetic field; RLC was chemically substituted by labeled RLC; axial orientational dynamics of the probe with respect to the muscle axis was determined. Chapter 4 utilizes 1) directional statistics that replaces the previous use of a Gaussian distribution and provides new insights into the degree of disorder and 2) a new bifunctional probe that adds an azimuthal dimension to the orientational data. Together, these techniques allow determination of the tilt and roll angles of the alpha helix without relying on the myosin structure.
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    Allostery governs Cdk2 activation and differential recognition of CDK inhibitors
    (2021-05) Majumdar, Abir
    Cyclin-dependent kinases (CDKs) are the master regulators of the eukaryotic cell cycle. To become activated, CDKs require both regulatory phosphorylation and binding of a cognate cyclin subunit. Using a series of DEER and NMR experiments, we studied the activation process of the G1/S kinase Cdk2 in solution. We show that catalytically inactive Cdk2 readily adopts multiple active-like states for efficient dephosphorylation, and that regulatory phosphorylation on the activation loop enhances allosteric coupling with the cyclin subunit. We then used DEER and FRET experiments to measure the binding of multiple CDK inhibitors and developed a thermodynamic model that describes the allosteric coupling between regulatory phosphorylation, cyclin binding and inhibitor binding. We reveal that the allosteric coupling between these biochemical effectors is responsible for the differential recognition of Cdk2 and Cdk4 inhibitors. Finally, we used sequence analysis, DEER, FRET and activity assays to identify and measure the effects of mutating an allosteric hub that has diverged between Cdk2 and Cdk4. We demonstrate that this hub controls the strength of allosteric coupling, and that the altered architecture and allosteric wiring of Cdk4 leads to compromised activity toward generic peptide substrates and comparative specialization toward its primary substrate retinoblastoma (RB).
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    Cardiac calcium transport regulation probed by electron paramagnetic resonance spectroscopy.
    (2010-07) Torgersen, Kurt Daniel
    Muscle contraction and relaxation is regulated by calcium flux between the sarcoplasmic reticulum and the cytoplasm. Subsequent to muscle contraction, calcium must be sequestered to the sarcoplasmic reticulum in order for muscle relaxation to occur. The sarco-endoplasmic reticulum Ca-ATPase (SERCA) is a P-type ATPase embedded in the SR membrane which uses ATP hydrolysis to pump calcium back into the SR lumen to facilitate muscle relaxation. In cardiac muscle, SERCA activity is regulated by phospholamban (PLB) a 52-residue integral membrane protein which exists in a dynamic equilibrium between monomeric and pentameric species. Previous data have shown that monomeric PLB is the primary regulator of SERCA activity but recent publications have proposed that the PLB pentamer may also bind to and inhibit SERCA activity. This inhibition can be relieved by phosphorylation of PLB at Ser16, although the mechanism is not known. Electron Paramagnetic Resonance (EPR) experiments were designed to test two proposed models of the PLB pentamer, the pinwheel and bellflower. Dynamics data using the TOAC amino acid spin label showed that, like the monomer, the pentamer is in a dynamic equilibrium between ordered (T) and dynamically disordered (R) states, with the T state being predominant. Accessibility of spin labels attached to the cytoplasmic domain to the lipid bilayer showed that, like the monomer, the pentamer cytoplasmic domains strongly interact with the lipid bilayer surface. Finally, pulsed EPR (DEER) experiments measuring long range distances between spin labels attached to the cytoplasmic domain showed a bimodal distance distribution with centers at 3 and 5 nm. All of these data support the pinwheel model. To investigate SERCA binding and phosphorylation affects on PLB dynamics, a monomeric mutant, AFA-PLB, was spin labeled with TOAC either the 11 position in the cytoplasmic domain or 36 in the transmembrane domain. Conventional EPR measurements showed that phosphorylation induced and order-to-disorder conformational change in the cytoplasmic domain and that SERCA preferentially binds the PLB R state. Phosphorylation of SERCA bound PLB resulted in a disorder-to-order conformational change, suggesting that pPLB is still bound to SERCA. Conventional dynamics from 36-TOAC in the transmembrane indicated a stable helix which was unaffected by phosphorylation or SERCA binding. Dipolar EPR measurements revealed that phosphorylation of PLB in the absence of SERCA induces oligomerization and that SERCA destabilizes the pPLB oligomer. Saturation Transfer EPR data which measures the rotational diffusion of PLB in the lipid bilayer supported the conclusion that phosphorylation of PLB in the absence of SERCA induces oligomerization and showed directly that phosphorylated PLB is still bound to active SERCA. These data support the model that phosphorylation dependent relief of SERCA inhibition does not require dissociation of the SERCA-PLB complex, but is rather the result of a structural change in the complex.
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    Elucidating the structural dynamics of SERCA-PLB regulation by electron paramagnetic resonance
    (2014-08) McCaffrey, Jesse Earl
    Muscle contraction and relaxation is initiated by changes in intracellular calcium, making adequate calcium transport essential for proper muscle function. A primary calcium transporter is the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA), located within the muscle cell and embedded in an organelle called the sarcoplasmic reticulum (SR). To facilitate muscle relaxation, calcium is sequestered from the cellular cytosol into the SR by SERCA. In cardiac muscle, enhanced regulation of calcium transport is needed to accommodate β-adrenergic stimulation (adrenaline demand), which is provided by the regulatory protein phospholamban (PLB). PLB binds to SERCA and inhibits calcium transport through reduction in calcium affinity. However, a phosphate group can be attached to PLB during adrenaline response (phosphorylation) which relieves PLB's inhibitory effect. The structural mechanisms for SERCA regulation by PLB, particularly with respect to phosphorylation, are not well-resolved. Under the Dissociation Model, PLB phosphorylation relieves SERCA inhibition by dissociating the SERCA-PLB complex. In contrast, the Subunit Model proposes that SERCA inhibition is relieved by a subtle structural change, where the SERCA-PLB complex is preserved. The primary goal of my thesis work is to elucidate the structural mechanisms of the SERCA-PLB complex using electron paramagnetic resonance (EPR) spectroscopy. The first study (Chapter 3) aims to discriminate between the Dissociation and Subunit models by measuring changes in the rotational diffusion of EPR spin labels rigidly coupled to PLB and SERCA. The second study (Chapter 4) further develops an anisotropic membrane system called bicelles for EPR orientation measurements on PLB and SERCA. The third study (Chapter 5) uses a combination of oriented bicelles and a novel rigid spin label (bifunctional spin label, or BSL) to measure PLB topology in the lipid membrane, with comparison to previous structural measurements by NMR and x-ray crystallography. Ongoing studies (Chapter 6) reconstitute both spin-labeled PLB and SERCA in bicelles to make PLB orientation measurements by EPR, as affected by PLB phosphorylation and binding of SERCA.
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    Kinetic and spectroscopic studies of cobalt- and manganese-substituted extradiol-cleaving homoprotocatechuate 2,3-dioxygenases
    (2013-02) Fielding, Andrew Jay
    Homoprotocatechuate (HPCA) 2,3-dioxygenase (HPCD) is an Fe(II)-dependent extradiol-cleaving dioxygenase, which oxidatively cleaves the aromatic C(2)-C(3) bond of its catecholic substrate. Here we compare the reactivity of Fe-HPCD with its Mn(II)- and Co(II)-substituted analogues. While Mn-HPCD exhibits steady-state kinetic parameters comparable to those of Fe-HPCD, Co-HPCD exhibits significantly higher KMO2 and kcat values. The high activity of Co-HPCD is surprising, given that cobalt has the highest standard M(III/II) redox potential of the three metals. These kinetic differences and the spectroscopic properties of Co-HPCD have proven to be useful in further exploring the unique O2 activation mechanism associated with the extradiol dioxygenase family. Employing the electron-poor substrate analogue 4-nitrocatechol (4NC), which is expected to slow down the rate of catechol oxidation, we were able to trap and characterize the initial O2-adduct in the single-turnover reaction of 4-nitrocatechol by Co-HPCD. This intermediate exhibits an S = 1/2 EPR signal typical of low-spin Co(III)−superoxide complexes. Both the formation and decay of the low-spin Co(III)−superoxide intermediate are slow compared to the analogous steps for turnover of 4NC by native high-spin Fe(II)-HPCD, which is likely to remain high-spin upon O2 binding. Possible effects of the observed spin-state transition upon the rate of O2 binding and catechol oxidation are discussed. Two transient intermediates were detected in the reaction of the [M-HPCD(4XC)] enzyme-substrate complexes (M = Mn or Co, and 4XC = 4-halocatechols, where X = F, Cl, and Br) with O2. The first intermediate (Co4XlCInt1) exhibited an S = 1/2 EPR signal associated with an organic radical species. Based on the UV-Vis and EPR data, Co4XCInt1 was assigned to a unique low-spin [Co(III)(4XSQ*)(hydro)peroxo] species where the semiquinone radical is localized onto C4 of the ring. M4XCInt2 was observed to have a high-spin metal(II) center by EPR and exhibit intense chromophores similar to the independently synthesized halogenated quinones (4XQ). Based on the UV-Vis and EPR data, M4XCInt2 is assigned to a [M(II)(4XQ)(hydro)peroxo] species. The M4XCInt2 species were further characterized by resonance Raman spectroscopy. Resonance enhanced vibrations between 1350-1450 cm-1 suggest that M4XCInt2 is a metal-semiquinone species, conflicting with the initial assignment of these intermediates as a quinone species. Based on the EPR and resonance Raman data, M4XCInt2 might be assigned to a [M(II)(SQ*)O2*-] diradical species.
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    Quantification of Wood Thermal Treatment by Electron Paramagnetic Resonance Spectroscopy
    (2020-12) McVay Jr., Jeffrey
    The process of thermally treating wood can impart desirable properties such as an aesthetically appealing color, dimensional stability, and increased resistance to moisture and fungal decay. However, potential undesirable outcomes, such as increased brittleness, may arise with extensive thermal treatment. Due to this risk, it is extremely important to quantify the extent of heat treatment on a specimen for quality control. Previously, free radical content in thermally treated wood was discovered. We therefore set out to quantify the radical content of thermally treated wood samples utilizing electron paramagnetic resonance (EPR) spectroscopy and attempted to find out if this could be correlated with the extent of heat treatment. We heat-treated samples using a pilot-scale, in-house oven kiln (an industrially relevant process), as well as in the highly controlled and quantifiable environment of a thermogravimetric analysis (TGA) analyzer. With these wood samples, we measured the free radical content using EPR to determine the correlation between radical content and other factors such as: treatment temperature, treatment atmosphere, rate of formation, treatment time, and the effects of moisture. Our main goal was to find reliable methods for quantifying the extent of heat treatment in thermally treated wood by the use of EPR spectroscopy.
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    SERCA-phospholamban structural dynamics studied by electron paramagnetic resonance
    (2013-07) James, Zachary Matthew
    Cardiac muscle function is regulated by calcium, with the sarcoplasmic reticulum (SR) serving as a calcium reservoir that releases its contents into the cytoplasm to initiate contraction. Relaxation requires that calcium be removed from the cytoplasm, which is accomplished primarily by the SR calcium-ATPase (SERCA), a transmembrane pump protein that consumes ATP to transport calcium to the SR interior, priming the muscle cell for another round of contraction. Cardiac SERCA is regulated by a second transmembrane SR protein, phospholamban (PLB), which binds and inhibits the ATPase unless phosphorylated at Ser16 by protein kinase A (PKA). The mechanism of SERCA inhibition by PLB, and the manner in which Ser16 phosphorylation reverses this inhibition remain poorly understood. The generally accepted model holds that calcium and PLB binding to SERCA are mutually exclusive, and that phosphorylation dissociates PLB to restore SERCA's calcium sensitivity. However, a number of spectroscopic studies have called this dissociation model into question, and instead suggest an alternative mechanism where PLB behaves as a subunit of SERCA, and changes its interactions with the ATPase upon phosphorylation to relieve inhibition.The work presented in this thesis tests several aspects of this alternative regulatory model, the Subunit Model, using spin-labeling and electron paramagnetic resonance (EPR) spectroscopy. In the first study, we used EPR to determine whether the inhibitory domain of PLB, its lone transmembrane helix, remains associated with SERCA following Ser16 phosphorylation. We found that the transmembrane helix of phosphorylated PLB remains bound to SERCA, in support of the Subunit Model, with our results hinting at a subtle structural change induced by phosphorylation. In the second study, we investigated the role of PLB's dynamic cytoplasmic helix in SERCA regulation, which equilibrates between an ordered T state and a dynamically disordered R state. Using charged lipids, we perturbed the electrostatic interactions between PLB's positively-charged cytoplasmic helix and the lipid bilayer, and then used EPR to resolve the T and R populations. We found that perturbation of the T/R equilibrium by charged lipids directly correlated with the functional state of SERCA, indicating that T and R correspond to inhibitory and non-inhibitory PLB conformations within the SERCA-PLB complex. In our final and ongoing study, we returned to PLB's transmembrane domain and used EPR accessibility measurements to detect conformational changes of this helix induced by Ser16 phosphorylation. Our results suggest that phosphorylation induces a subtle change in the tilt and/or rotation of PLB's transmembrane helix relative to SERCA, which may break inhibitory interactions and restore SERCA calcium sensitivity without dissociating the regulatory complex.
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    Structural Dynamics of the Calmodulin-Ryanodine Receptor Interaction Using Bifunctional Spin Labels and EPR
    (2018-08) Her, Cheng
    Muscle contraction and relaxation are regulated by changes in intracellular calcium levels. To facilitate muscle contraction, calcium is released from the intracellular calcium reservoir into the cytosol by the homotetrameric calcium channel known as the ryanodine receptor (RyR). The sarcoplasmic reticulum membrane-embedded RyR is a target for many small molecule and protein modulators, including the ubiquitously expressed calcium binding protein calmodulin (CaM). CaM can bind four calcium ions via its four EF-hand motifs and has calcium-dependent effects on RyR. It is well established that CaM potentiates channel opening below µM calcium and inhibition above µM calcium. Despite this, the structural mechanism of the calcium-dependent CaM-mediated RyR regulation remain poorly understood. The primary goal of the work presented here is to elucidate the structural mechanisms of the CaM-RyR interaction, using bifunctional spin labels and electron paramagnetic resonance (EPR). In the first study, we investigated the structural dynamics of a spin labeled ryanodine receptor peptide (RyRp) bound to CaM using EPR (Chapter 4). By detecting the rotational dynamics of specific sites along the backbone, we show that the interaction of RyRp with CaM is nonuniform along the peptide, and the primary effect of calcium is to increase the interaction of the N-lobe of CaM with RyRp. In the second study (Chapter 5), we placed spin probes on both CaM and RyRp and investigated the calciumdependent structural changes of the complex using a distance measurement EPR technique known as double electron-electron resonance (DEER). Our DEER distance results provide support for the conformational selection mechanism of CaM binding to RyRp (i.e. the binding of RyRp shifts CaM to preexisting structural states). We discovered differential Ca effects on the two lobes of CaM with respect to RyRp binding. More specifically, we discovered that Ca was required for complete interaction of the N-lobe with RyRp, while the C-lobe bound RyRp independent of Ca. These findings are consistent with results from Chapter 4 and provide support for the hypothesis that CaM functions as a subunit of RyR through binding of the C-lobe, and complete interaction of the N-lobe of CaM (in response to increased cytosolic Ca levels) is responsible for maximum inhibition of RyR. Thus, our results provide novel insight into the structural mechanism of CaM-mediated RyR regulation while showcasing an innovative approach with wide applicability to other biological systems.
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    Structural transitions of myosin associated with force generation in spin-labeled muscle fibers.
    (2012-06) Mello, Ryan Nicholas
    Muscle contraction is driven by the actin-activated hydrolysis of ATP by myosin, resulting in the relative sliding of actin and myosin filaments. Current models propose that filament sliding is driven by a structural transition of myosin’s catalytic domain (CD) and light chain domain (LCD). The goal of this research is to measure structural transitions of myosin II (muscle and nonmuscle) that are associated for force generation. Structural measurements were made using electron paramagnetic resonance (EPR) spectroscopy. This work is comprised of two separate, but related, projects. In the first project (Chapter 3), thiol crosslinking and EPR were used to resolve structural transitions of myosin’s LCD and CD that are associated with force generation. Spin labels were incorporated into the LCD of muscle fibers by exchanging spin-labeled regulatory light chain (RLC) for endogenous RLC, with full retention of function. LCD orientation and dynamics were measured in three biochemical states: relaxation (A.M.T), post-hydrolysis intermediate (A.M.D.P), and rigor (A.M.D). To trap myosin in a structural state analogous to the elusive post-hydrolysis ternary complex A.M.D.P, we used pPDM to crosslink SH1 (Cys707) to SH2 (Cys697) on the CD. EPR showed that the LCD of crosslinked fibers has an orientational distribution intermediate between relaxation and rigor, and saturation transfer EPR revealed slow rotational dynamics indistinguishable from that of rigor. Similar results were obtained for the CD using a bifunctional spin label to crosslink SH1 to SH2, but the CD was more disordered than the LCD. We conclude that SH1-SH2 crosslinking traps a state in which both the LCD and CD are in a structural state intermediate between relaxation (highly disordered and microsecond dynamics) and rigor (highly ordered and rigid), supporting the hypothesis that the crosslinked state is an A.M.D.P analog on the force generation pathway. In the second project, we present a method for obtaining high-resolution structural information of proteins using EPR of a bifunctional spin label (BSL). Two complimentary EPR techniques were employed to measure dynamics and orientation (conventional EPR) and intraprotein distances (dipolar electron-electron resonance). The exploitation of BSL is a key feature of this work. BSL attaches at residue positions i and i+4, which drastically restricts probe motion compared to monofunctional probes. For comparison, measurements were also made with the monofunctional spin label MSL. Subfragment 1 of Dictyostelium myosin II (S1dC) was used to exemplify the increased resolution provided by BSL. Using this approach, we demonstrate with experiments that BSL significantly increases resolution when measuring distance and orientation compared to MSL. And while this work does focus on the methodology, there is significant biological insight into myosin’s nucleotide-dependent structural transitions.