Time-resolved surface charge change on the cytoplasmic side of bacteriorhodopsin.
The pH-sensitive dye 5-iodoacetamidofluorescein was covalently bound to a single cysteine residue introduced by site-directed mutagenesis in position 101 on the cytoplasmic surface or in position 130 on the extracellular surface of the proton pump bacteriorhodopsin. Using time-resolved absorption spectroscopy at 495 nm a transient increase was observed in the apparent pK of the dye attached at residue 101. At pH 7.3 the rise and decay times of this pK-change (approximately 2 ms and approximately 60 ms) correlate well with decay times observed for the M and O intermediates and with the proton uptake time. Interpreting the pK-increase of +0.18 pH-unit in terms of a transiently more negative surface charge density, we calculate a change of -0.80 elementary charge per bacteriorhodopsin at the cytoplasmic surface. It is likely that this charge change is due to the transient deprotonation of aspartate-96. With the label in position 130 on the extracellular surface no transient pK-shift was detected.
Alexiev
U
U
Biophysics Group, Freie Universität Berlin, Germany.
Scherrer
P
P
Marti
T
T
Khorana
H G
HG
Heyn
M P
MP
D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D004134:Dimyristoylphosphatidylcholine; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D011199:Potentiometry; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods; D013997:Time Factors
D011994:Recombinant Proteins; D001436:Bacteriorhodopsins; D004134:Dimyristoylphosphatidylcholine