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Time‐resolved surface charge change on the cytoplasmic side of bacteriorhodopsin
Author(s) -
Alexiev U.,
Scherrer P.,
Marti T.,
Khorana H.G.,
Heyn M.P.
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)00985-i
Subject(s) - bacteriorhodopsin , cytoplasm , charge (physics) , biophysics , chemistry , chemical physics , physics , biology , biochemistry , membrane , particle physics
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 (∼2 ms and ∼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.