TIME-RESOLVED SURFACE-CHARGE CHANGE ON THE CYTOPLASMIC SIDE OF BACTERIORHODOPSIN

被引:22
作者
ALEXIEV, U [1 ]
SCHERRER, P [1 ]
MARTI, T [1 ]
KHORANA, HG [1 ]
HEYN, MP [1 ]
机构
[1] MIT,DEPT BIOL & CHEM,CAMBRIDGE,MA 02139
关键词
SURFACE CHARGE DENSITY; GOUY-CHAPMAN THEORY; TRANSIENT SURFACE POTENTIAL CHANGES; BACTERIORHODOPSIN; PHOTOCYCLE;
D O I
10.1016/0014-5793(95)00985-I
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 (similar to 2 ms and similar to 60 ms) correlate well with decay times observed for the M and O intermediates and with the proton uptake time. Interpreting the pg-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.
引用
收藏
页码:81 / 84
页数:4
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