Sergeev Y V, Hejtmancik J F, Wingfield P T
National Eye Institute, National Institutes of Health, Bethesda, Maryland 20982, USA.
Biochemistry. 2004 Jan 20;43(2):415-24. doi: 10.1021/bi034617f.
Beta-crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for lens clarity and refraction. They undergo modification as the lens ages, including cleavage of their terminal extensions. The energetics of betaA3- and betaB2-crystallin association was studied using site-directed mutagenesis and analytical ultracentrifugation. Recombinant (r) murine wild type betaA3- and betaB2-crystallins were modified by removal of either the N-terminal extension of betaA3 (rbetaA3Ntr) or betaB2 (rbetaB2Ntr), or both the N- and C-terminal extensions of betaB2 (rbetaB2NCtr). The proteins were expressed in Sf9 insect cells or Escherichia coli and purified by gel-filtration and ion-exchange chromatography. All beta-crystallins studied demonstrated fast reversible monomer-dimer equilibria over the temperature range studied (5-35 degrees C) with a tendency to form tighter dimers at higher temperatures. The N-terminal deletion of rbetaA3 (rbetaA3Ntr) significantly increases the enthalpy (+10.9 kcal/mol) and entropy (+40.7 cal/deg mol) of binding relative to unmodified protein. Removal of both N- and C-terminal extensions of rbetaB2 also increases these parameters but to a lesser degree. Deletion of the betaB2-crystallin N-terminal extension alone (rbetaB2Ntr) gave almost no change relative to rbetaB2. The resultant net negative changes in the binding energy suggest that betaAlpha3- and betaB2-crystallin association is entropically driven. The thermodynamic consequences of the loss of betaAlpha3-crystallin terminal extensions by in vivo proteolytic processing could increase their tendency to associate and so promote the formation of higher order associates in the aging and cataractous lens.
β-晶状体蛋白是哺乳动物晶状体的主要蛋白质成分，其稳定性以及形成高阶复合物的能力对于晶状体的透明度和折射至关重要。随着晶状体老化，它们会发生修饰，包括末端延伸部分的裂解。利用定点诱变和分析型超速离心法研究了βA3-和βB2-晶状体蛋白缔合的能量学。通过去除βA3（rβA3Ntr）或βB2（rβB2Ntr）的N端延伸部分，或同时去除βB2的N端和C端延伸部分（rβB2NCtr），对重组（r）小鼠野生型βA3-和βB2-晶状体蛋白进行修饰。这些蛋白质在Sf9昆虫细胞或大肠杆菌中表达，并通过凝胶过滤和离子交换色谱法进行纯化。在所研究的温度范围（5-35℃）内，所有研究的β-晶状体蛋白均表现出快速可逆的单体-二聚体平衡，且在较高温度下倾向于形成更紧密的二聚体。相对于未修饰的蛋白质，rβA3（rβA3Ntr）的N端缺失显著增加了结合的焓（+10.9千卡/摩尔）和熵（+40.7卡/度摩尔）。去除rβB2的N端和C端延伸部分也会增加这些参数，但程度较小。单独缺失βB2-晶状体蛋白的N端延伸部分（rβB2Ntr）相对于rβB2几乎没有变化。结合能产生的净负变化表明βA3-和βB2-晶状体蛋白的缔合是由熵驱动的。体内蛋白水解过程导致βA3-晶状体蛋白末端延伸部分丧失的热力学后果可能会增加它们缔合的倾向，从而促进老化和白内障晶状体中高阶缔合体的形成。
