Subunit exchange of lens alpha-crystallin: a fluorescence energy transfer study with the fluorescent labeled alphaA-crystallin mutant W9F as a probe.
Subunit exchange of lens alpha-crystallin: a fluorescence energy transfer study with the fluorescent labeled alphaA-crystallin mutant W9F as a probe.
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晶状体α-晶状体蛋白的亚基交换：以荧光标记的αA-晶状体蛋白突变体W9F作为探针的荧光能量转移研究。
DOI：
10.1016/s0014-5793(98)00707-8
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发表时间：
1998
期刊：
影响因子：
3.5
通讯作者：
Liang,JJ
中科院分区：
文献类型：
--
作者：
Sun,TX;Akhtar,NJ;Liang,JJ
通过定点突变制备无Trp αA-晶状体蛋白突变体（W 9 F）。该突变体在构象（二级和三级结构）方面似乎与野生型相同。用巯基特异性荧光探针2-（4′-马来酰亚胺苯胺基）萘-6-磺酸盐（MIANS）标记W 9 F，并用于αA-和αA-晶体蛋白之间以及αA-和αB-晶体蛋白之间的亚基交换，通过测量荧光共振能量转移进行研究。当发生亚基交换时，在野生型αA-或αB-晶状体蛋白的Trp（供体，最大发射波长为336 nm）和标记W 9 F的MIANS（受体，最大吸收波长为313 nm）之间观察到能量转移。记录Trp的时间依赖性降低和MIANS荧光的增加。在37°C下的交换比在25°C下更快。同质亚基之间的能量转移效率（αA-αA）大于异质亚基之间的能量转移效率（αA-αB）。先前的等电聚焦交换研究表明αA和αB亚基之间存在完全但缓慢的交换。本研究表明，交换是一个快速的过程，αA-αA和αA-αB之间不同的能量转移效率表明，αA-和αB-晶体蛋白不一定是结构等效的。
A Trp‐free αA‐crystallin mutant (W9F) was prepared by site‐directed mutation. This mutant appears to be identical to the wild‐type in terms of conformation (secondary and tertiary structures). W9F was labeled with a sulfhydryl‐specific fluorescent probe, 2‐(4′‐maleimidylanilino) naphthalene‐6‐sulfonate (MIANS), and used in a subunit exchange between αA‐ and αA‐crystallins as well as between αA‐ and αB‐crystallins, studied by measurement of fluorescence resonance energy transfer. Energy transfer was observed between Trp (donor, with emission maximum at 336 nm) of wild‐type αA‐ or αB‐crystallin and MIANS (acceptor, with absorption maximum at 313 nm) of labeled W9F when subunit exchange occurred. Time‐dependent decrease of Trp and increase of MIANS fluorescence were recorded. The exchange was faster at 37°C than at 25°C. The energy transfer efficiency was greater between homogeneous subunits (αA‐αA) than between heterogeneous subunits (αA‐αB). A previous exchange study with isoelectric focusing indicated a complete but slow exchange between αA and αB subunits. The present study showed that the exchange was a fast process, and the different energy transfer efficiencies between αA‐αA and αA‐αB indicated that αA‐ and αB‐crystallins were not necessarily structurally equivalent.