Scam feels the pinch.
Scam feels the pinch.
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DOI：
10.1085/jgp.117.3.235
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发表时间：
2001-03
期刊：
影响因子：
--
通讯作者：
Karlin A
中科院分区：
文献类型：
--
作者：
Karlin A
Movileanu 等人 (2001) 在本期杂志上发表的论文描述了替代半胱氨酸可及性方法（SCAM；Akabas 等人，1992；Akabas 等人，1994）对于定位通道管腔最窄区域问题的适用性测试。 Movileanu 等人 (2001) 使用异常大的硫氢基定向试剂来探测葡萄球菌溶血素形成的宽孔通道。该蛋白质的膜相关形式的结构已得到高分辨率解析（Song 等人，1996）。它是一种蘑菇形七聚体复合物，插入易感细胞的膜中，其宽帽位于细胞外侧或人工平面脂质膜的顺侧，并且茎穿过双层（参见 Movileanu 等人，2001 年的图 1）。细胞外端或顺端的孔入口宽 30 Å。孔立即变窄至 20 Å，然后变宽，形成 46 Å 宽的大前庭。孔再次变窄至 16 Å，这是其主要收缩部分，然后继续延伸到其细胞内或反端，形成不规则的圆柱体，宽度约为 20 Å。 Bayley 和同事 (Movileanu et al., 2001) 证明，他们可以通过应用 SCAM 和足够大的试剂来定位孔的最窄收缩处。这些试剂是平均分子量为 1,000、1,800、2,500 和 5,000 D 的聚乙二醇 (PEG) 2-吡啶基二硫化物衍生物。它们与半胱氨酸反应形成混合二硫化物，其中 PEG 部分连接到半胱氨酸硫上。如果这些试剂是未水合的球体，它们的直径将为 16、19、21 和 27 Å；除第一个外，所有孔都太大而无法通过溶血素孔。然而，细长构型一定是普遍存在的，因为除了 5-kD 试剂之外的所有试剂都穿过孔。通过偶联转录和翻译在体外合成半胱氨酸取代的溶血素突变体、纯化并掺入人工平面双层中。通过半胱氨酸与 PEG 试剂对孔电导的影响来监测其反应，这种影响通过清洗不可逆，但通过还原可逆。定位通道最窄收缩的基础是，在所有其他条件相同的情况下，添加到膜一侧的试剂与收缩近侧的半胱氨酸的反应速率将比与收缩远侧的半胱氨酸的反应速率更快。然而，所有其他因素很少相同。许多因素可以影响孔中半胱氨酸的反应速率（Wilson 和 Karlin，2001）。有两种过程：（1）将试剂转移到半胱氨酸附近或从半胱氨酸附近转移，这决定了试剂的局部浓度； (2) 局部试剂与半胱氨酸的反应（Pascual 和 Karlin，1998b）。转移速率常数取决于半胱氨酸路径上的空间位阻和静电，而局部反应速率常数取决于局部空间位阻和静电。这里考虑的与巯基吡啶衍生物的反应，就像与甲硫代磺酸盐衍生物的反应一样，仅与去质子化的半胱氨酸硫氢基发生（Roberts等人，1986）。因此，局部反应速率取决于巯基 pKa 和局部 pH 值，这也取决于局部静电相互作用。
The paper by Movileanu et al.(2001) in this issue of The Journal describes a test of the applicability of the substituted-cysteine-accessibility method (SCAM; Akabas et al., 1992; Akabas et al., 1994) to the problem of locating the narrowest region of a channel lumen. Movileanu et al.(2001) use unusually large sulfhydryldirected reagents to probe the wide bore channel formed by Staphylococcal-hemolysin. The structure of the membrane-associated form of this protein has been solved to high resolution (Song et al., 1996). It is a mushroom-shaped heptameric complex that inserts in the membrane of a susceptible cell with the wide cap on the extracellular side, or on the cis side of an artificial planar lipid membrane, and with the stem traversing the bilayer (see Figure 1 in Movileanu et al., 2001). The entrance to the pore at the extracellular or cis end is 30 Å wide. The pore immediately narrows to 20 Å, and then widens into a large vestibule 46 Å across. The pore again narrows to 16 Å, its major constriction, and then continues to its intracellular or trans end as an irregular cylinder, which is roughly 20 Å wide. Bayley and co-workers (Movileanu et al., 2001) demonstrate that they can locate the narrowest constriction of the pore by applying SCAM with large enough reagents. The reagents were 2-pyridyl disulfide derivatives of polyethylene glycol (PEG) of average molecular masses: 1,000, 1,800, 2,500 and 5,000 D. They react to form mixed disulfides with cysteine in which the PEG moiety is attached to the cysteine sulfur. If these reagents were unhydrated spheres, their diameters would be 16, 19, 21, and 27 Å; all but the first too large to pass through the-hemolysin pore. However, elongated configurations must be prevalent because all but the 5-kD reagent pass through the pore. Cysteine-substituted-hemolysin mutants were synthesized in vitro by coupled transcription and translation, purified, and incorporated into artificial planar bilayers. The reactions of the cysteines with the PEG reagents were monitored by their effects on the conductance of the pore, which were irreversible by washing but reversible by reduction. The basis for locating the narrowest constriction of the channel is simply that, all other things being equal, the rate of reaction of a reagent added to one side of the membrane with a cysteine on the near side of the constriction will be faster than the rate of reaction with a cysteine on the far side of the constriction. However, all other things are seldom equal.Numerous factors can influence the reaction rate of a cysteine in a pore (Wilson and Karlin, 2001). There are two kinds of processes:(1) the transfer of reagent to and from the vicinity of the cysteine, which determines the local concentration of the reagent; and (2) the reaction of local reagent with the cysteine (Pascual and Karlin, 1998b). The transfer rate constants depend on steric hindrance and electrostatics along the pathway to the cysteine, whereas the local reaction rate constant depends on local steric hindrance and electrostatics. The reaction considered here with mercaptopyridine derivatives, like the reaction with methanethiosulfonate derivatives, takes place exclusively with the deprotonated cysteine sulfhydryl (Roberts et al., 1986). Hence, the local reaction rate depends on the sulfhydryl pKa and the local pH, which also depends on local electrostatic interactions.
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影响因子：
16.2
作者：
Larsson, HP;Baker, OS;Isacoff, EY
通讯作者：
Isacoff, EY
影响因子：
3.4
作者：
KIRSCH, GE;ALAM, M;HARTMANN, HA
通讯作者：
HARTMANN, HA
影响因子：
16.2
作者：
Liu, Y;Jurman, ME;Yellen, G
通讯作者：
Yellen, G
影响因子：
16.2
作者：
Liu, Y;Holmgren, M;Yellen, G
通讯作者：
Yellen, G
DOI：
10.1085/jgp.111.6.717
发表时间：
1998-06
期刊：
The Journal of general physiology
影响因子：
--
作者：
Pascual JM;Karlin A
通讯作者：
Karlin A