Stopped-flow analysis on anion binding to blue-form halorhodopsin from Natronobacterium pharaonis: comparison with the anion-uptake process during the photocycle.
Pharaonis halorhodopsin (phR), the light-driven chloride ion pump from Natronobacterium pharaonis with C-terminal histidine tag, was expressed in Escherichia coli cells. The protein was solubilized with 0.1% n-dodecyl beta-D-maltopyranoside and purified with a nickel column. Removal of Cl- from the medium yields blue phR (phR(blue)) that has lost Cl- near the chromophore. Addition of Cl- converts phR(blue) to a red-shifted Cl--bound form (phR(Cl)). Circular dichroic spectra of phR(blue) and phR(Cl) exhibited a bilobe in the visual region, indicating specific oligomerization of the phR monomers. The order of anion concentration which induced a shift from phR(blue) to phR(X) was Br- < Cl- < NO3- < N3-, which was the same as in the case of phR purified from N. pharaonis membranes. Chloride binding kinetics was measured by time-resolved absorption changes with stopped-flow rapid mixing. Rates of Cl- binding consisted of fast and slow components, and the amplitude of the fast component was about 90% of the total changes. The rate constant of the fast component at 100 mM NaCl at 25 degrees C was 260 s(-1) with an apparent activation energy of 35 kJ/mol. These values are in good agreement with the process of Cl- uptake in the photocycle (O --> hR' reaction) reported previously [Váró et al. (1995) Biochemistry 34, 14500-14507]. In addition, the Cl- concentration dependence on both rates was similar to each other. These observations suggest that the O-intermediate is similar to phR(blue) and that Cl- uptake during the photocycle may be ruled by a passive process.
Sato
Maki
M
Division of Biological Sciences, Graduate School of Science, and Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan.
Kanamori
Tatsuaki
T
Kamo
Naoki
N
Demura
Makoto
M
Nitta
Katsutoshi
K
D000838:Anions / Q000378:metabolism*; D019843:Archaeal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002338:Carotenoids / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D025602:Halorhodopsins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006639:Histidine / Q000235:genetics; D007700:Kinetics; D008027:Light; D019633:Natronobacterium / Q000737:chemistry*; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D025603:Sensory Rhodopsins; D013053:Spectrophotometry; D017186:Titrimetry
D000838:Anions; D019843:Archaeal Proteins; D025602:Halorhodopsins; D011994:Recombinant Proteins; D025603:Sensory Rhodopsins; C061508:sensory rhodopsin II protein, archaeal; D002338:Carotenoids; D006639:Histidine