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Structural Studies of Soluble Guanylyl Cyclase and Its Bacterial Homologs

MA, Xiaolei
http://rave.ohiolink.edu/etdc/view?acc_num=case1208379599

Abstract Details

2008, Doctor of Philosophy, Case Western Reserve University, Biochemistry.
The most well known receptor for NO is soluble guanylate cyclase (sGC), which converts guanosine-5'-triphosphate (GTP) to cyclic guanosine-3', 5'-monophosphate (cGMP) upon NO activation. cGMP serves as an important second messenger to regulate a wide range of physiological process in cardiovascular and neurological systems. This pathway is targeted for therapeutic purposes as Glyceryl trinitrate (GTN), which generates the signaling molecule NO inside the cell, has been used to treat angina pectoris and heart failure for more than a century. sGC is a heterodimeric hemoprotein composed of two different subunits: α (73-82 kDa) and β (70-76 kDa). Both subunits share a similar domain organization: an N-terminal domain (which harbors a heme only in β1 called H-NOX), a central H-NOXA domain, a Coiled-coil (CC) domain and a C-terminal guanylyl cyclase domain. An appreciation of the atomic details of the mechanism by which NO activates sGC is critical to our understanding of how such a small gaseous molecule NO is able to recognized by the target cell and in turn elicit a wide variety of responses. This thesis is focused on the structural characterization of H-NOX domain, H-NOXA domain and CC domain, little structural information was available by the time this project was initiated. The first target was the stand-alone H-NOX domain from Nostoc sp PCC 7120, which shares 33% sequence identity with human sGCβ1. We have determined three Ns H-NOX structures including unliganded, NO bound and CO bound form at 2.1A, 2.5A and 2.6A respectively. We have identified a critical aromatic residue above the heme plane conferring binding advantage to NO over CO by steric hindrance. Comparison of these structures and previously published Tt H-NOX structure has revealed a heme pivot-bend mechanism that correlates with the H-NOX structural changes with respect to their presumed activation state. The second target was the H-NOXA domain from a STHK gene in the Nostoc punctiforme PCC 73102 genome, which is 35% identical to human sGCβ1 H-NOXA domain. We have determined the Np STHK N-terminal H-NOXA domain in a dimerized form at 2.1A. The monomer adopts a PAS-like fold and dimerizes in a similar fashion as observed in other PAS dimers by juxtapositioning of the N-terminal helices with their preceding residues and the face of the β-sheet. We proposed sGC H-NOXA domain could hetero-dimerize in the same fashion and we confirmed our hypothesis by Ala scanning mutagenesis studies. The structural information provides broad implications including PAS-mediated dimerization for preferential heterodimerization of sGC, allosteric regulation of sGC and Np STHK, and sGC domain organization. The third target was the CC domain from rat sGC. We have obtained single crystals for this domain and successfully prepared selenomethionine substituted crystals for phasing. However due to the presence of pseudo merohedral twinning in both of the crystal forms, structural determination and refinement is still in progress.
Menachem Shoham, PhD (Committee Chair)
Focco van den Akker, PhD (Advisor)
Paul Carey, PhD (Committee Member)
Irene Lee, PhD (Committee Member)
Saurav Misra, PhD (Committee Member)
102 p.
Biochemistry; Biophysics
sGC; H-NOX; H-NOXA; Heme; STHK; Ns H-NOX

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Citations

  • MA, X. (2008). Structural Studies of Soluble Guanylyl Cyclase and Its Bacterial Homologs [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1208379599

    APA Style (7th edition)

  • MA, Xiaolei. Structural Studies of Soluble Guanylyl Cyclase and Its Bacterial Homologs. 2008. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1208379599.

    MLA Style (8th edition)

  • MA, Xiaolei. "Structural Studies of Soluble Guanylyl Cyclase and Its Bacterial Homologs." Doctoral dissertation, Case Western Reserve University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=case1208379599

    Chicago Manual of Style (17th edition)

case1208379599
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© 2008, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.