Probing the sensory rhodopsin II binding domain of its cognate transducer by calorimetry and electrophysiology.
Sensory rhodopsin II, a repellent phototaxis receptor from Natronobacterium pharaonis (NpSRII) forms a tight complex with its cognate transducer (NpHtrII). Light excitation of the receptor triggers conformational changes in both proteins, thereby activating the cellular two-component signalling cascade. In membranes, the two proteins form a 2:2 complex, which dissociates to a 1:1 heterodimer in micelles. Complexed to the transducer sensory rhodopsin II is no longer capable of light-driven proton pumping. In order to elucidate the dimerisation and the size of the receptor-binding domain of the transducer, isothermal titration calorimetry and electrophysiological experiments have been carried out. It is shown, that an N-terminal sequence of 114 amino acid residues is sufficient for tight binding (K(d)=240nM; DeltaH=-17.6kJmol(-1)) and for inhibiting the proton transfer. These data and results obtained from selected site-directed mutants indicate a synergistic interplay of transducer transmembrane domain (1-82) and cytoplasmic peptide (83-114) leading to an optimal and specific interaction between receptor and transducer.
Hippler-Mreyen
Silke
S
Department of Physical Biochemistry, Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany.
Klare
Johann P
JP
Wegener
Ansgar A
AA
Seidel
Ralf
R
Herrmann
Christian
C
Schmies
Georg
G
Nagel
Georg
G
Bamberg
Ernst
E
Engelhard
M
M
D000818:Animals; D019843:Archaeal Proteins / Q000737:chemistry*; D002151:Calorimetry; D002338:Carotenoids / Q000737:chemistry*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D025602:Halorhodopsins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008823:Micelles; D008958:Models, Molecular; D009865:Oocytes; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011522:Protons; D012333:RNA, Messenger / Q000378:metabolism; D025603:Sensory Rhodopsins; D015398:Signal Transduction; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014981:Xenopus
D019843:Archaeal Proteins; D025602:Halorhodopsins; D008823:Micelles; D010455:Peptides; D011522:Protons; D012333:RNA, Messenger; D025603:Sensory Rhodopsins; C061508:sensory rhodopsin II protein, archaeal; D002338:Carotenoids