Sun T X, Akhtar N J, Liang J J
Center for Ophthalmic Research, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
FEBS Lett. 1998 Jul 3;430(3):401-4. doi: 10.1016/s0014-5793(98)00707-8.
A Trp-free alphaA-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 alphaA- and alphaA-crystallins as well as between alphaA- and alphaB-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 alphaA- or alphaB-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 degrees C than at 25 degrees C. The energy transfer efficiency was greater between homogeneous subunits (alphaA-alphaA) than between heterogeneous subunits (alphaA-alphaB). A previous exchange study with isoelectric focusing indicated a complete but slow exchange between alphaA and alphaB subunits. The present study showed that the exchange was a fast process, and the different energy transfer efficiencies between alphaA-alphaA and alphaA-alphaB indicated that alphaA- and alphaB-crystallins were not necessarily structurally equivalent.
通过定点突变制备了一种无色氨酸的αA-晶状体蛋白突变体（W9F）。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'-马来酰亚胺基苯胺基）萘-6-磺酸盐（MIANS）标记W9F，并将其用于αA-晶状体蛋白与αA-晶状体蛋白之间以及αA-晶状体蛋白与αB-晶状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-晶状体蛋白的色氨酸（供体，发射最大值在336nm）与标记的W9F的MIANS（受体，吸收最大值在313nm）之间观察到能量转移。记录了色氨酸荧光随时间的下降和MIANS荧光的增加。在37℃时交换比在25℃时更快。同型亚基（αA-αA）之间的能量转移效率高于异型亚基（αA-αB）之间的能量转移效率。先前用等电聚焦进行的交换研究表明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过程
