Fluorescence energy transfer studies on the proximity of the two essential thiols of myosin subfragment-1.
Fluorescence energy transfer studies on the proximity of the two essential thiols of myosin subfragment-1.
复制标题
肌球蛋白亚片段 1 两个必需硫醇接近度的荧光能量转移研究。
DOI：
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发表时间：
1983
影响因子：
4.8
通讯作者：
F. Garland
中科院分区：
文献类型：
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作者：
H. Cheung;F. Gonsoulin;F. Garland
5-（碘乙酰氨基乙基）氨基萘-1-磺酸通过连接到另一个的受体5-（碘乙酰氨基）荧光素共价连接到一个硫醇。从受体敏化的测量中获得了可比较的结果。当供体与SH 1连接，受体与SH 2连接时，基于两个连接探针的各向同性和快速随机取向的假设的平均供体-受体分离（R（2/ 3））为46 A。由于这两种探针的取向自由度部分受到限制，R的下限和上限由观察到的转移数据和由受体和受体标记的蛋白质确定的去极化因子计算。平均下限（R（min））为29，平均上限（R（max））为76。在这三个距离参数中，R（2/3）和R（max）可以基于结构理由而被排除，因为已知两个巯基在重链的一级结构中仅相距10个残基。当供体和受体位置被切换时，这些参数的值稍小。加入F-肌动蛋白使R（min）降低5 A，而MgADP对最小分离没有可检测的影响。用稳态和纳秒方法研究了丙烯酰胺对施主发射的猝灭。得到的证据表明，发射的共价连接的供体包含两个组件。丙烯酰胺的存在促进了寿命的解析。这种发射的猝灭归因于碰撞猝灭。当供体位于SH 1巯基时，MgADP降低了猝灭，但当供体连接到SH 2位点时，MgADP对猝灭没有影响。连接到SH 1的供体也被碘化物猝灭，并且猝灭模式与多个锡永组分相容。
5-(iodoacetamidoethyl)aminonaphthalene-l-sulfonic acid covalently attached to one thiol by the acceptor 5- (iodoacetamido)fluorescein linked to the other. Com- parable results were obtained from measurements of acceptor sensitization. When the donor was attached to SH1 and the acceptor to SH2, the mean donor-acceptor separation based on the assumption of isotropic and rapid random orientation of both attached probes (R(2/ 3)) is 46 A. Since the orientational freedom of both probes was partially restricted, lower and upper bounds of R were calculated from the observed transfer data and depolarization factors determined from do- nor- and acceptor-labeled proteins. The mean lower bound (R(min)) is 29 and the mean upper bound (R(max)) is 76 A. Of these three distance parameters, R(2/3) and R(max) may be ruled out on structural grounds since the two thiols are known to be only 10 residues apart in the primary structure of the heavy chain. Slightly smaller values of these parameters were obtained when the donor and acceptor locations were switched. Addition of F-actin reduced R(min) by 5 A, whereas MgADP had no detectable effect on the mini- mum separation. Quenching of the donor emission by acrylamide was investigated by both the steady state and nanosecond methods. Evidence was obtained that the emission of the covalently attached donor contained two components. Resolution of the lifetimes was facil- itated by the presence of acrylamide. The quenching of this emission is attributed to collisional quenching. MgADP reduced the quenching when the donor was located at the SH1 thiol, but it had no effect on the quenching when attached to the SH2 site. The donor attached to SH1 was also quenched by iodide and the quenching pattern was compatible with multiple emis- sion components.