Browsing by Subject "Recombinant Proteins"
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Item Biochemical Analysis of Apoptosome Formation(2006-12-19) Kim, Hyun-Eui; Abrams, JohnApoptosis is an active cell death program executed by proteases named caspases. One of the major caspase-activating pathways is initiated by mitochondria. Upon various apoptotic stimuli, the mitochondria releases cytochrome c into the cytosol where it binds to apoptotic protease activating factor 1 (Apaf-1). Then, the cytochrome c-bound Apaf-1 forms a heptameric complex named apoptosome. Apoptosome provides a platform to activate downstream caspases. The initiator caspase, procaspase-9 is recruited to apoptosome and gets activated to cleave downstream effector caspases. The series of this activation cascade is tightly regulated at each step. However, the role of cytochrome c and nucleotides during apoptosome formation is not clear. Also, how the apoptosome activity is stimulated by the positive regulator PHAP proteins is yet to be determined. Thus, my thesis work includes studies regarding these questions using biochemical analysis. I reconstituted apoptosome pathway using recombinant proteins in vitro. As a result, I discovered several biochemical steps during apoptosome formation that were previously unknown. And I identified a new mediator that positively regulates apoptosome formation. The new findings are, 1) Recombinant Apaf-1 obtained from insect cell expression system was already associated with dATP. 2) The Apaf-1-bound dATP got hydrolyzed upon cytochrome c binding. 3) The hydrolyzed nucleotide on Apaf-1 needed to be exchanged with dATP/ATP to form an active apoptosome. 4) CAS is a mediator of PHAP proteins. PHAPI and CAS enhanced the nucleotide exchange on Apaf-1 to stimulate apoptosome formation.Item Structural Insights into Ion Selectivity and Calcium Blockage in Cyclic Nucleotide Gated Channels(2010-07-12T18:56:12Z) Derebe, Mehabaw Getahun; Jiang, YouxingCyclic nucleotides-gated (CNG) channels play an essential role in the visual and olfactory sensory systems and are ubiquitously expressed in a variety of neuronal and non neuronal cells. Details of their underlying ion selectivity properties are still not fully understood and a matter of debate in the absence of high resolution structures. Presented in this study are high resolution (1.58-1.95Å) crystal structures and functional analyses of engineered mimics of CNG channels by duplicating their selectivity filter sequences in the background of the bacterial non-selective NaK channel. Mimics share several striking functional similarities in ion selectivity with eukaryotic CNG channels: they are non-selective and permeate Na+ and K+ equally well; externally added Ca2+ serves as a permeating blocker, with the conserved acidic residue in the filter mediating Ca2+ binding. Structures reveal a hitherto unseen selectivity filter architecture that suggests that CNG channel selectivity filters likely comprise three contiguous ion binding sites. The high resolution structures also allow for a thorough characterization of monovalent and divalent ion permeation which, in combination with electrophysiological recordings, offers structural insight into CNG channel function at an unprecedented level of detail.