Liang Jack J, Liu Bing-Fen
Ophthalmic Research/Surgery, Brigham and Women's Hospital, Department of Ophthalmology, Harvard Medical School, Boston, MA 02115, USA.
Protein Sci. 2006 Jul;15(7):1619-27. doi: 10.1110/ps.062216006. Epub 2006 Jun 2.
Lens alpha-crystallin is an oligomeric protein with a molecular mass of 500-1000 kDa and a polydispersed assembly. It consists of two types of subunits, alphaA and alphaB, each with a molecular mass of 20 kDa. The subunits also form homo-oligomers in some other tissues and in vitro. Their quaternary structures, which are dynamic and characterized by subunit exchange, have been studied by many techniques, including fluorescence resonance energy transfer (FRET) and mass spectrometry analysis. The proposed mechanism of subunit exchange has been either by dissociation/association of monomeric subunits or by rapid equilibrium between oligomers and suboligomers. To explore the nature of subunit exchange further, we performed additional FRET measurements and analyses using a fluorescent dye-labeled W9F alphaA-crystallin as the acceptor probe and Trp in other crystallins (wild-type and R116C alphaA, wild-type and R120G alphaB, wild-type and Q155* betaB2) as the donor probe and calculated the transfer efficiency, Förster distance, and average distance between two probes. The results indicate only slight decreased efficiency and increased distance between two probes for the R116C alphaA and R120G alphaB mutations despite conformational changes.
晶状体α-晶体蛋白是一种寡聚蛋白，分子量为500-1000 kDa，组装呈多分散性。它由两种亚基组成，αA和αB，每个亚基的分子量为20 kDa。这些亚基在其他一些组织和体外也会形成同型寡聚体。它们的四级结构具有动态性，其特征是亚基交换，已经通过包括荧光共振能量转移（FRET）和质谱分析在内的多种技术进行了研究。提出的亚基交换机制要么是单体亚基的解离/缔合，要么是寡聚体和亚寡聚体之间的快速平衡。为了进一步探索亚基交换的本质，我们使用荧光染料标记的W9F αA-晶体蛋白作为受体探针，以其他晶体蛋白（野生型和R116C αA、野生型和R120G αB、野生型和Q155* βB2）中的色氨酸作为供体探针，进行了额外的FRET测量和分析，并计算了转移效率、福斯特距离和两个探针之间的平均距离。结果表明，尽管构象发生了变化，但R116C αA和R120G αB突变的两个探针之间的效率仅略有下降，距离增加。
