Pyrene excimer fluorescence in rabbit skeletal alphaalphatropomyosin labeled with N-(1-pyrene)maleimide. A probe of sulfhydryl proximity and local chain separation.
Pyrene excimer fluorescence in rabbit skeletal alphaalphatropomyosin labeled with N-(1-pyrene)maleimide. A probe of sulfhydryl proximity and local chain separation.
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用 N-(1-芘)马来酰亚胺标记的兔骨骼 α-肌球蛋白中的芘准分子荧光。
DOI：
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发表时间：
1978
影响因子：
4.8
通讯作者：
S. Lehrer
中科院分区：
文献类型：
--
作者：
S. Betcher;S. Lehrer
使用荧光试剂 N-(1-芘)马来酰亚胺在半胱氨酸 190 处特异性标记兔骨骼 α-原肌球蛋白。通过在 pH 6.0 (PyrI-alphaalphaTm) 或 pH 7.5 (PyrII-alphaalphaTm) 下标记获得光谱上不同的产品。 PyrII-alphaalphaTm 是由 PyrI-alphaalphaTm 的半胱氨酸 190 处的 N-(1-芘)琥珀酰亚胺部分和同一条链上的赖氨酸基团（可能是赖氨酸 189）之间的二次反应产生的。芘准分子荧光存在于天然状态中，但在两种产物的未折叠状态中不存在，从而验证了 -SH 基团和链配准模型的接近性 原肌球蛋白的结构。对 PyrII-ααTm 的氯化胍依赖性解折叠的研究表明，在解折叠之前准分子荧光的丧失，为半胱氨酸 190 附近的分子中优先不稳定的区域提供了证据。这项工作表明，如果存在两个或多个，N-(1-芘)马来酰亚胺可用于探测任何蛋白质中的 SH 邻近度和局部构象。 怀疑近端--SH 群体。
Rabbit skeletal alphaalphatropomyosin was specificially labeled at cysteine 190 with the fluorescent reagent, N-(1-pyrene)maleimide. Spectroscopically different products were obtained by labeling at pH 6.0 (PyrI-alphaalphaTm) or pH 7.5 (PyrII-alphaalphaTm). PyrII-alphaalphaTm results from a secondary reaction between the N-(1-pyrene)succinimido moiety at cysteine 190 of PyrI-alphaalphaTm and a lysine group on the same chain, probably lysine 189. Pyrene excimer fluorescence was present in the native state but absent in the unfolded state of both products, thus verifying the proximity of the--SH groups and the chain register model for the structure of tropomyosin. Studies of the guanidinium chloride-dependent unfolding of PyrII-alphaalphaTm showed that loss of excimer fluorescence precedes unfolding, providing evidence for a region of preferential instability in the molecule near cysteine 190. This work suggests that N-(1-pyrene)maleimide could be used to probe both--SH proximity and local conformation in any protein if the presence of two or more proximal--SH groups is suspected.