Browsing by Subject "DEER"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    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).
  • Thumbnail Image
    Item
    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.
  • Thumbnail Image
    Item
    Structural dynamics of the myosin force-generating region.
    (2009-12) Agafonov, Roman
    Myosin is a molecular motor that generates force on actin using energy from ATP hydrolysis. Myosin plays a key role in muscle contraction and is responsible for a variety of motility processes at the cellular level. It works cyclically, changing its conformation during the power stroke and the recovery stroke. X-ray crystallography has provided information about the structural organization of myosin in different biochemical states (as defined by bound nucleotide), inspiring several structural models that could explain the molecular mechanism of myosin's function. Spectroscopy, in combination with site-directed labeling and transient experiments, can test and refine these models and provide information about myosin's dynamic properties. The goal of this project was to determine the structural dynamics of the myosin force-generating domain and study coupling mechanisms between this domain and the myosin active site. We have chosen Dictyostelium discoideum (Dicty) as our experimental system since it provides multiple advantages in comparison with the muscle myosin. In particular, it is possible to manipulate the Dicty DNA sequence, engineering labeling sites at desired locations and introducing functional mutations at the points of interest. As a first part of the project, we have tested Dicty myosin in comparison with myosin purified from rabbit skeletal muscle, and have shown that structural changes in the force-generating domain of Dicty and rabbit myosin are identical. We then focused on specific elements within the force-generating domain, relay helix and relay loop, as these elements appeared to be crucial for interdomain coupling and force generation. Using time-resolved EPR and FRET, we have developed a spectroscopic approach to determine the conformation of the relay helix. We have also developed a novel technique that we called transient time-resolved FRET [(TR)2FRET], which allowed us to monitor structural changes within the relay helix in real time. We then studied the relationship between the state of the myosin active site (which is determined by the bound ligand) and the structure of the relay helix. To obtain insights about regulatory mechanisms, we have investigated the effect of a mutation that is known to abolish myosin motor function, despite leaving enzymatic activity intact. These experiments revealed important coupling mechanisms between the relay loop and relay helix, providing a structural explanation for the previously observed functional effects and a model for power stroke activation in myosin.