Covalently bound pH-indicator dyes at selected extracellular or cytoplasmic sites in bacteriorhodopsin. 1. Proton migration along the surface of bacteriorhodopsin micelles and its delayed transfer from surface to bulk.
Covalently bound pH-indicator dyes at selected extracellular or cytoplasmic sites in bacteriorhodopsin. 1. Proton migration along the surface of bacteriorhodopsin micelles and its delayed transfer from surface to bulk.
复制标题
在细菌视紫红质中选定的细胞外或细胞质位点共价结合 pH 指示剂染料。
DOI：
10.1021/bi00250a019
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Heyn,MP
中科院分区：
文献类型：
--
作者：
Scherrer,P;Alexiev,U;Marti,T;Khorana,HG;Heyn,MP
1994年8月25日收到的修订版Mandalpt ®摘要：光诱导的质子释放和吸收的动力学是用与细菌视紫红质的细胞外和细胞质表面上的不同位点共价结合的光学pH指示剂荧光素监测的。选择性标记是通过（碘乙酰氨基）荧光素与细菌视紫红质中的单个半胱氨酸残基反应实现的，所述半胱氨酸残基通过定点诱变引入到所需位置。所有的测量都是在22 ℃、pH7.3、150 mMKCl中用磷脂/去污剂混合物中的细菌视紫红质胶束进行的.无论是半胱氨酸的替代品，也没有随后的标记影响的吸收光谱的细菌视紫红质和上升时间的M中间体。只有衰变的M改变了一些细菌视紫红质突变体的半胱氨酸残基的细胞质侧。用附着在细胞外表面（质子释放侧）72位（在连接螺旋B和C的环中）或130位（DE环）的荧光素检测到的质子释放时间为22±4 ps，明显快于在水相中用吡喃测得的质子释放时间（野生型和所有研究的突变体为125±10 ps）。对于在细胞质环区域（质子摄取侧）中的位置35、101、160、229和231处标记的细菌视紫红质突变体，在61± 4ps的时间内观察到释放的质子。这比细胞外侧的释放时间慢约3倍，但仍显著快于在本体相中用吡喃测量的释放时间。这些结果表明，释放的质子保留在胶束表面上，并更迅速地沿着这个表面的细胞质侧比从表面到散装介质。这一结论得到了实验的支持，其中质子迁移率沿着胶束表面的变化，通过添加磷脂与不同pKfs的头基的细菌视紫红质/CHAPS胶束。在细胞质侧上具有标记的情况下，当添加DMPC（p <2.2;质子停留时间ss 10 ns）或DMPA（p <8.0;质子停留时间<<10 ms）时，光诱导的瞬时质子化变化的幅度分别增加或降低。然而，在细胞外侧用指示剂未检测到磷脂的影响。这些观察更快的质子扩散沿着胶束表面比他们的平衡从表面到散装支持efficientproton耦合沿质子源和汇之间的膜表面的生物能模型。我们的数据还表明，与细胞外细菌视紫红质（bR）1是盐生盐杆菌紫膜中的一种七螺旋跨膜蛋白，具有光驱动质子泵的功能[综述如下，参见Stoeckenius和Bogomolni（1982），Lanyi（1992），Oesterhelt等人，（1992）和Rothschild（1992）]。与视色素一样，bR含有亚视黄基发色团（Oesterhelt & Stoeckenius，1971），其具有与Lys-216连接的质子化视网膜席夫碱（刘易斯et al.，1974年）。当
Revised Manuscript Received August 25, 1994® abstract: The kinetics of the light-induced release and uptake of protons was monitored with the optical pH-indicator fluorescein covalently boundto various sites on the extracellular and cytoplasmic surfaces of bacteriorhodopsin. Selective labeling was achieved by reacting (iodoacetamido) fluorescein with the single cysteine residues in bacteriorhodopsin introduced at the desiredpositions by site-directed mutagenesis. All measurements were performed with bacteriorhodopsin micelles in phospholipid/detergent mixtures in 150 mM KC1 at 22 C, pH 7.3. Neither the replacements by cysteine nor the subsequent labeling affected the absorption spectrum of bacteriorhodopsin and the rise times of the M intermediate. Only the decay of M was altered for some bacteriorhodopsin mutants with cysteine residues on the cytoplasmic side. The proton release time detected with fluorescein attachedto the extracellular surface (the proton release side) at position 72 (in the loop connecting helices B and C) or 130 (DE loop) was 22±4 ps, clearly faster than that measured with pyranine in the aqueous bulk phase (125±10 ps for wild-type and all mutants studied). For bacteriorhodopsin mutants labeled at positions 35, 101, 160, 229, and 231 in the cytoplasmic loop region (the proton uptake side), the released proton was observed with a time of 61±4 ps. This was about 3-fold slower than the release time on the extracellular side, but still significantly faster than that measured with pyranine in the bulk phase. These results suggest that the released protons are retained on the micellar surface and move more rapidly along this surface to the cytoplasmic side than from the surface to the bulk medium. This conclusion is supported by experiments in which the proton mobility along the micellar surface was varied by adding phospholipids with headgroups of different pKfs to the bacteriorhodopsin/CHAPS micelles. With the label on the cytoplasmic side, the amplitude of the light-induced transient protonation change increased or decreased, respectively, when DMPC (p£ 2.2; protondwell time ss 10 ns) or DMPA (pÁ" 8.0; proton dwell time « 10 ms) was added. However, no effect of the phospholipids was detected with the indicator on the extracellular side. These observations of faster proton diffusion along the micellar surface than their equilibration from the surface to the bulk support bioenergetic models of efficientproton coupling alongthe membrane surface between proton sources and sinks. Our data also demonstrate that a probe bound to the extracellular (proton release) surface is required to detect the actual appearance of the pumped proton on the protein surfaceand to correlate it with a specific photocycle intermediate.Bacteriorhodopsin (bR) 1 is a seven helical transmembrane protein in the purple membrane of Halobacterium salinarium and functions as a light-driven protonpump [for a review, see Stoeckenius and Bogomolni (1982), Lanyi (1992), Oesterhelt et al.(1992), and Rothschild (1992)]. Like visual pigments, bR contains a retinylidene chromophore (Oesterhelt & Stoeckenius, 1971) with a protonated retinal Schiff base linkage to Lys-216 (Lewis et al., 1974). When