Application of ANS fluorescent probes to identify hydrophobic sites on the surface of DREAM.
DREAM (calsenilin or KChIP-3) is a calcium sensor involved in regulation of diverse physiological processes by interactions with multiple intracellular partners including DNA, Kv4 channels, and presenilin, however the detailed mechanism of the recognition of the intracellular partners remains unclear. To identify the surface hydrophobic surfaces on apo and Ca(2+)DREAM as a possible interaction sites for target proteins and/or specific regulators of DREAM function the binding interactions of 1,8-ANS and 2,6-ANS with DREAM were characterized by fluorescence and docking studies. Emission intensity of ANS-DREAM complexes increases upon Ca(2+) association which is consistent with an overall decrease in surface polarity. The dissociation constants for ANS binding to apoDREAM and Ca(2+)DREAM were determined to be 195±20μM and 62±4μM, respectively. Fluorescence lifetime measurements indicate that two ANS molecules bind in two independent binding sites on DREAM monomer. One site is near the exiting helix of EF-4 and the second site is located in the hydrophobic crevice between EF-3 and EF-4. 1,8-ANS displacement studies using arachidonic acid demonstrate that the hydrophobic crevice between EF-3 and EF-4 serves as a binding site for fatty acids that modulate functional properties of Kv4 channel:KChIP complexes. Thus, the C-terminal hydrophobic crevice may be involved in DREAM interactions with small hydrophobic ligands as well as other intracellular proteins.
Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA.
Walter G
WG
Gonzalez
Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA. Electronic address: miksovsk@fiu.edu.
Jaroslava
J
Miksovska
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry*; D000818:Animals; D016718:Arachidonic Acid / Q000737:chemistry; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D051605:Kv Channel-Interacting Proteins / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D062105:Molecular Docking Simulation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics
D000817:Anilino Naphthalenesulfonates; C496756:Csen protein, mouse; D005456:Fluorescent Dyes; D051605:Kv Channel-Interacting Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; C047136:2-anilinonaphthalene-6-sulfonic acid; D016718:Arachidonic Acid; C027132:1-anilino-8-naphthalenesulfonate; D002118:Calcium
ANS; Calcium signaling; Calsenilin; EF-hand; KChIP; Neuronal calcium sensor