Real time analysis of interaction between inositol 1,4, 5-trisphosphate receptor type I and its ligand.
Inositol 1,4,5-trisphosphate (IP(3)) is an important second messenger that releases intracellular Ca(2+) by binding to its specific receptor, inositol 1,4,5-trisphosphate receptor (IP(3)R), in a wide range of cellular processes. We report here large-scale expression and purification of N-terminal 604 amino acids of IP(3)R type 1 (T604) expressed in E. coli, which contains the ligand binding domain. Surface plasmon resonance biosensor studies showed that purified T604 could bind to its ligands with binding specificity identical to that of full-length native IP(3)R type 1. Kinetic parameters of T604 for IP(3) consisted of a fast association rate constant (K(ass) = 1.2 x 10(6) M(-1) s(-1)) and a rapid dissociation rate constant (k(diss) = 1 s(-1)), and the equilibrium dissociation constant was determined to be 336 nM, at 150 mM NaCl and pH 7.4. However, association and dissociation patterns depended on the pH level and ionic strength. These results pave the way toward detail analysis of structure-function analysis of the ligand binding domain of IP(3)R type 1 for its ligands.
Natsume
T
T
Calciosignal Net Project, Exploratory Research for Advanced Technology (ERATO) JST., c/o RIKEN Komagome Branch, 2-28-8, Honkomagome, Bunkyo-ku, 113-0021, Japan. natsumw@ims.u-tokyo.ac.jp
Hirota
J
J
Yoshikawa
F
F
Furuichi
T
T
Mikoshiba
K
K
D000595:Amino Acid Sequence; D015220:Calcium Channels / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007700:Kinetics; D008024:Ligands; D009994:Osmolar Concentration; D011485:Protein Binding; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins