Clarke M J, Artero J B, Moulin M, Callow P, Carver J A, Griffiths P C, Haertlein M, Harding J J, Meek K M, Timmins P, Regini J W
School of Optometry and Vision Sciences, Cardiff University, Maindy Road, Cathays, Cardiff, CF24 4LU, UK.
Biochim Biophys Acta. 2010 Mar;1800(3):392-7. doi: 10.1016/j.bbagen.2009.12.001. Epub 2009 Dec 11.
alpha-Crystallin, one of the main constituent proteins in the crystalline lens, is an important molecular chaperone both within and outside the lens. Presently, the structural relationship between alpha-crystallin and its target proteins during chaperone action is poorly understood. It has been hypothesised that target proteins bind within a central cavity. Small-angle neutron-scattering (SANS) experiments in conjunction with isotopic substitution were undertaken to investigate the interaction of a target lens protein (gammaE-crystallin) with alpha-crystallin (alpha(H)) and to measure the radius of gyration (Rg) of the proteins and their binary complexes in solution under thermal stress. The size of the alpha(H) in D(2)O incubated at 65 degrees C increased from 69+/-3 to 81+/-5 A over 40 min, in good agreement with previously published small-angle X-ray scattering (SAXS) and SANS measurements. Deuterated gammaE-crystallin in H(2)O buffer (gammaE(D)/H(2)O) and hydrogenous gammaE-crystallin in D(2)O buffer (gammaE(H)/D(2)O) free in solution were of insufficient size and/or too dilute to provide any measurable scattering over the angular range used, which was selected primarily to investigate gammaE:alpha(H) complexes. The evolution of the aggregation size/shape as an indicator of alpha(H) chaperone action was monitored by recording the neutron scattering in different H:D solvent contrasts under thermally stressed conditions (65 degrees C) for binary mixtures of alpha(H), gammaE(H), and gammaE(D). It was found that Rg(alpha(H):gammaE(D)/D(2)O)>Rg(alpha(H):gammaE(H)/D(2)O)>Rg(alpha(H)/D(2)O) and that Rg(alpha(H):gammaE(H)/D(2)O) approximately Rg(alpha(H)/D(2)O). The relative sizes observed for the complexes weighted by the respective scattering powers of the various components imply that gammaE-crystallin binds in a central cavity of the alpha-crystallin oligomer, during chaperone action.
α-晶状体蛋白是晶状体中的主要组成蛋白之一，在晶状体内外都是一种重要的分子伴侣。目前，人们对α-晶状体蛋白与其靶蛋白在伴侣作用过程中的结构关系了解甚少。据推测，靶蛋白结合在一个中央腔内。进行了小角中子散射（SANS）实验并结合同位素取代，以研究一种靶晶状体蛋白（γE-晶状体蛋白）与α-晶状体蛋白（α（H））的相互作用，并测量热应激条件下蛋白质及其二元复合物在溶液中的回转半径（Rg）。在65℃下孵育的D₂O中的α（H）大小在40分钟内从69±3增加到81±5 Å，这与先前发表的小角X射线散射（SAXS）和SANS测量结果非常吻合。溶液中游离的H₂O缓冲液中的氘代γE-晶状体蛋白（γE（D）/H₂O）和D₂O缓冲液中的含氢γE-晶状体蛋白（γE（H）/D₂O）尺寸不足和/或过于稀释，无法在所用的角度范围内提供任何可测量的散射，该角度范围主要用于研究γE:α（H）复合物。通过记录α（H）、γE（H）和γE（D）二元混合物在热应激条件（65℃）下不同H:D溶剂对比度下的中子散射，监测聚集尺寸/形状的演变作为α（H）伴侣作用的指标。发现Rg（α（H）:γE（D）/D₂O）>Rg（α（H）:γE（H）/D₂O）>Rg（α（H）/D₂O），且Rg（α（H）:γE（H）/D₂O）≈Rg（α（H）/D₂O）。由各组分各自的散射能力加权得到的复合物相对大小表明，在伴侣作用过程中，γE-晶状体蛋白结合在α-晶状体蛋白寡聚体的中央腔内。
