This project investigates how chemical toxins or physical factors alter metabolic processes. NMR methods provide a unique approach for the investigation of metabolic and physiological processes in intact systems, perfused organs, cell suspensions, as well as by examination of cell extracts. The main studies performed as part of this research effort during the past year are summarized below: Project 1: We have been interested in determining whether the peroxymonocarbonate ion, which forms spontaneously from the interaction of peroxide with carbon dioxide, may play a role in the peroxidase activity of superoxide dismutase (SOD). During the past year, kinetic evidence was obtained in support of the role of HCO4- ion as an oxidant for reduced Cu,Zn-SOD. The formation of a carbonate radical anion as a result of this interaction would then lead to further oxidative reactions. By using conventional stopped-flow spectrophotometry, rate constants consistent with such a mechanism were obtained. Structural analyis of the SOD suggests that the dissociation of His61 from the active site Cu(I) in SOD-Cu(I) can contribute to this chemistry by facilitating the binding of larger anions, such as peroxymonocarbonate. Project 2. Orai1 is a protein responsible for calcium influx from the extracellular environment into cytosol following calcium depletion of the endoplasmic reticulum (ER), a process called store-operated calcium entry (SOCE). Upon calcium depletion, the plasma membrane protein Orai1 is activated by the ER membrane protein STIM1, resulting in an increase of cytosolic Ca2+. Calcium concentration in the cytosol is strictly maintained at a low level. This requires rapid inactivation of Orai1 after stimulation, a process called calcium dependent inactivation (CDI). It has been shown that calmodulin inactivates Orai1 by binding to an N-terminal fragment called calmodulin binding domain (CMBD). The interaction between Orai1 and calmodulin at the atomic level has not been characterized. As part of this project, we have worked with the Birnbaumer group to characterize the crystal and solution characteristics of this interaction. Crystallographic evidence demosntrates that the Orai-CMBD interacts with the C-terminal domain of calmodulin, but simultaneous binding interactions with the N-terminal domain are not observed. However, isothermal titration calorimetry demonstrates binding of the Orai-CMBD to both lobes of calmoduin, with the affinity for the N-terminal lobe about five times weaker than for the C-terminal lobe. Solution NMR studies demonstrate that the Orai-CMBD does indeed bind to both lobes of the calmodulin, and is capable of forming a 2:1 CMBD-calmodulin complex. Furthermore, the interactions with each lobe are homologous. Thus, both lobes of calmodulin compete for the same binding residues of the Orai-CMBD, consistent with the absence of a tight 1:1 complex involving simultaneous binding to both lobes of calmodulin. This study for the first time quantifies the affinity of Orai1 to calmodulin. We propose that calmodulin inactivates Orai1 by reducing the affinity of STIM1 for Orai1.

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Wang, Huanchen; DeRose, Eugene F; London, Robert E et al. (2014) IP6K structure and the molecular determinants of catalytic specificity in an inositol phosphate kinase family. Nat Commun 5:4178
Leinisch, Fabian; Jiang, Jinjie; DeRose, Eugene F et al. (2013) Investigation of spin-trapping artifacts formed by the Forrester-Hepburn mechanism. Free Radic Biol Med 65:1497-1505
Odet, Fanny; Gabel, Scott; London, Robert E et al. (2013) Glycolysis and mitochondrial respiration in mouse LDHC-null sperm. Biol Reprod 88:95
Ranguelova, Kalina; Ganini, Douglas; Bonini, Marcelo G et al. (2012) Kinetics of the oxidation of reduced Cu,Zn-superoxide dismutase by peroxymonocarbonate. Free Radic Biol Med 53:589-94
McCormack, Thomas; Petrovich, Robert M; Mercier, Kelly A et al. (2010) Identification and functional characterization of a novel acetylcholine-binding protein from the marine annelid Capitella teleta. Biochemistry 49:2279-87
Bonini, Marcelo G; Gabel, Scott A; Ranguelova, Kalina et al. (2009) Direct magnetic resonance evidence for peroxymonocarbonate involvement in the cu,zn-superoxide dismutase peroxidase catalytic cycle. J Biol Chem 284:14618-27
Gabel, Scott A; London, Robert E (2008) Ternary borate-nucleoside complex stabilization by ribonuclease A demonstrates phosphate mimicry. J Biol Inorg Chem 13:207-17