Browsing by Subject "GTP Phosphohydrolases"
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Item FRET-Based Conformational Sensor for the m1 Muscarinic Cholinergic Receptor(2012-07-20) Chang, Seungwoo; Ross, Elliott M.Signaling behaviors of G-protein-mediated signaling are the outcome of a regulated cycle of GTP binding and hydrolysis. Binding of GTP, which activates G proteins, is promoted by an agonist-activated receptor; and hydrolysis of bound GTP, which deactivates G proteins, is accelerated by a GTPase-activating protein (GAP). These two processes need to be coordinately regulated to achieve fast turning on and off of signaling with robust signal output. I developed an optical conformational sensor for the m1 muscarinic cholinergic receptor (M1), a prototypical G protein-coupled receptor (GPCR), to study how receptor activity is regulated by the coordinated action of agonist, G protein and GAP. To create the sensor, I adopted an underlying design originally developed by the Lohse group. The sensor exploits intramolecular fluorescence resonance energy transfer, FRET, to monitor activation-associated conformational changes in intracellular loop 3 of the receptor. In the sensor, a CFP FRET donor and a labeling site for FlAsH (fluorescein-based biarsenical dye) FRET acceptor are engineered into the M1 receptor at the C terminus and loop 3, respectively. The development proceeded through several distinct optimization steps that probably reflect general considerations for developing such sensors for class A GPCRs. After optimizing the labeling conditions to approach stoichiometric derivatization by FlAsH, I found that the fluorescence response of the sensor depended on: (1) the location of the FlAsH labeling site in loop 3; (2) the length of the C-terminal region, which apparently acts as a lever arm, prior to placement of the CFP; and (3) the choice among circularly permuted CFP moieties. Finally, based on a homology-modeled structure of the M1 receptor, placement of the FlAsH site and the length of its flexible linkers were re-optimized to prevent interference with binding of the sensor to G?q. The sensor retained essentially wild-type agonist binding and signaling activity of the M1 receptor in living cells and cell membranes. Fluorescence responses of the sensor to muscarinic agonists paralleled their cellular efficacies. The sensor in living cells faithfully reported agonist-driven conformational change of the M1 receptor. Therefore, the FRET-based sensor proves to be a useful tool to investigate the mechanisms by which conformational dynamics of the M1 receptor is regulated by agonist in living cells and membranes. Effects of G?q on the conformation of the M1 receptor could not be determined because stable interaction between the M1 receptor and G?q could not be detected in cells or cell membranes either by fluorescence change or by agonist binding affinity; this is also true for wild-type receptor. Although the sensor reconstituted in phospholipid vesicles retained wild-type agonist binding and signaling function, fluorescence response to agonist was not detected in the vesicles. I demonstrated that solubilization of the sensor denatured a substantial fraction of the sensor, resulting in low fractional ligand binding activity of in vitro labeled sensor and thus artifactually low fluorescence response to agonist.Item Mechanistic Analysis of Microtubule Dynamics and Regulation(2018-10-15) Geyer, Elisabeth Anne; Jaqaman, Khuloud; Rosen, Michael K.; Yu, Hongtao; Rice, Luke M.Microtubules are critical components in a cells cytoskeletal network, known to form the mitotic spindle which allows for chromosome segregation and cell division and organizing the cytoplasm of non-dividing cells. The quick reorganization of the cytoskeleton relies heavily on the underlying behavior of microtubules, known as dynamic instability. Dynamic instability, the rapid switch between growing and shrinking states of the microtubule, depends on the functional GTPase behavior of the microtubule polymerizing subunits, αβ-tubulin. Recent studies have noted the presence of multiple conformational states of αβ-tubulin in the microtubule lattice, in addition to major conformational changes that occur in αβ-tubulin within the microtubule as compared to free αβ-tubulin in solution. In Chapter 2, I will discuss a study in which I explored the role of the conformational cycle and its impact on microtubule dynamic instability. By studying a mutation in β-tubulin, T238A, I have shown that nucleotide hydrolysis and conformational changes in the lattice are tightly linked and provide allostery throughout the microtubule. Uncoupling the two cycles disrupts the allostery which greatly impacts the rapid transitions normally seen in dynamic instability that allow for fast and decisive structural rearrangements. In Chapter 3, I will discuss a study that aimed to dissect the molecular mechanisms of the yeast microtubule polymerase, Stu2p. In this project, I developed an all-yeast in vitro reconstitution system using total internal reflection fluorescence microscopy which enabled me to study a variety of Stu2 mutants, in the presence of wild-type and mutant yeast tubulin samples. Here, I discovered how the tubulin conformational state can impact Stu2 function and determined a new property of Stu2 in its ability recognize and bind either the microtubule lattice or free tubulin. From these findings, I have proposed a new alternating engagement mechanism to explain how Stu2 functions processively at the microtubule plus end to increase the growth rates of microtubules. Finally, in Chapter 4 I will summarize my work on both of these projects and discuss both future directions and preliminary results looking to solve the structure of human β:T238A microtubules using cryo-EM.