Lindner R A, Kapur A, Mariani M, Titmuss S J, Carver J A
Department of Chemistry, The University of Wollongong, NSW, Australia.
Eur J Biochem. 1998 Nov 15;258(1):170-83. doi: 10.1046/j.1432-1327.1998.2580170.x.
The small heat-shock protein, alpha-crystallin, has chaperone ability whereby it stabilises proteins under stress conditions. In this study, alterations in the structure of alpha-crystallin during its interaction with a variety of substrate proteins (insulin, alpha-lactalbumin, ovotransferrin and serum albumin) under stress conditions have been examined using visible absorption, 31P-NMR and 1H-NMR and fluorescence spectroscopy. The fluorescence and 31P-NMR data imply that during the chaperone action of alpha-crystallin under reducing conditions, there is a slight increase in hydrophilicity of its N-terminal region and an alteration in flexibility of its C-terminal region, but overall, alpha-crystallin does not undergo a gross structural change. The fluorescence data suggest that substrate proteins interact with alpha-crystallin in a molten globule or intermediately folded state. The same conclusion is made from 1H-NMR spectroscopic monitoring of the interaction of alpha-crystallin with substrate proteins, e.g. the insulin B chain. The stoichiometry of interaction between alpha-crystallin and the various substrate proteins reveals that steric factors are important in determining the efficiency of interaction between the two proteins, i.e. on a molar subunit basis, alpha-crystallin is a more efficient chaperone protein with smaller substrate proteins. Comparison is also made between the high-molecular-mass (HMM) complexes formed between alpha-crystallin and ovotransferrin when reduced and heat stressed. Under heating conditions, fluorescence spectroscopy indicates that the HMM complex has a greater exposure of hydrophobicity to solution than that formed by reduction. Furthermore, in interacting with heated ovotransferrin, the C-terminal extension of the alphaB-crystallin subunit preferentially loses its flexibility suggesting that it is involved in stabilising bound ovotransferrin. By contrast, this extension is only partially reduced in flexibility in the HMM complex formed after reduction of ovotransferrin. The functional role of the C-terminal extensions in the chaperone action and the overall quaternary structure of alpha-crystallin is discussed.
小分子热休克蛋白α-晶体蛋白具有伴侣能力，可在应激条件下稳定蛋白质。在本研究中，利用可见吸收光谱、31P-NMR、1H-NMR和荧光光谱，研究了应激条件下α-晶体蛋白与多种底物蛋白（胰岛素、α-乳白蛋白、卵转铁蛋白和血清白蛋白）相互作用过程中其结构的变化。荧光和31P-NMR数据表明，在还原条件下α-晶体蛋白发挥伴侣作用时，其N端区域的亲水性略有增加，C端区域的柔韧性发生改变，但总体而言，α-晶体蛋白并未发生重大结构变化。荧光数据表明，底物蛋白以熔融球状体或中间折叠状态与α-晶体蛋白相互作用。通过对α-晶体蛋白与底物蛋白（如胰岛素B链）相互作用的1H-NMR光谱监测也得出了相同结论。α-晶体蛋白与各种底物蛋白之间相互作用的化学计量表明，空间因素在决定这两种蛋白之间相互作用的效率方面很重要，即从摩尔亚基角度来看，α-晶体蛋白对较小的底物蛋白是一种更有效的伴侣蛋白。还比较了α-晶体蛋白与卵转铁蛋白在还原和热应激条件下形成的高分子质量（HMM）复合物。在加热条件下，荧光光谱表明，HMM复合物比还原形成的复合物具有更大的疏水性暴露于溶液中。此外，在与加热的卵转铁蛋白相互作用时，αB-晶体蛋白亚基的C端延伸优先失去其柔韧性，表明它参与稳定结合的卵转铁蛋白。相比之下，在卵转铁蛋白还原后形成的HMM复合物中，这种延伸的柔韧性仅部分降低。讨论了C端延伸在α-晶体蛋白的伴侣作用和整体四级结构中的功能作用。
