Wijesinha-Bettoni R, Dobson C M, Redfield C
Oxford Centre for Molecular Sciences, University of Oxford, New Chemistry Laboratory South Parks Road, Oxford, OX1 3QT, UK.
J Mol Biol. 2001 Mar 30;307(3):885-98. doi: 10.1006/jmbi.2001.4530.
In the presence of 0.5 M NaCl at pH 7.1, the Ca(2+)-free apo form of recombinant bovine alpha-lactalbumin (BLA) is sufficiently stabilised in its native state to give well-resolved NMR spectra at 20 degrees C. The (1)H and (15)N NMR resonances of native apo-BLA have been assigned, and the chemical-shifts compared with those of the native holo protein. Large changes observed between the two forms of BLA are mainly limited to the Ca(2+)-binding region of the protein. These data suggest that Na(+) stabilises the native apo state through the screening of repulsive negative charges, at the Ca(2+)-binding site or elsewhere, rather than by a specific interaction at the vacant Ca(2+)-binding site. The hydrogen exchange protection of residues in the Ca(2+)-binding loop and the C-helix is reduced in the apo form compared to that in the holo form. This indicates that the dynamic behaviour of this region of the protein is substantially increased in the absence of the bound Ca(2+). Real-time NMR experiments show that the rearrangements of the structure associated with the conversion of the holo to apo form of the protein do not involve the detectable population of partially unfolded intermediates. Rather, the conversion appears to involve local reorganisations of the structure in the vicinity of the Ca(2+)-binding site that are coupled to the intrinsic fluctuations in the protein structure.
在pH 7.1的0.5 M NaCl存在下，重组牛α-乳白蛋白（BLA）的无钙脱辅基形式在其天然状态下得到充分稳定，能够在20℃下给出分辨率良好的NMR谱。已对天然脱辅基BLA的（1）H和（15）N NMR共振进行了归属，并将化学位移与天然全蛋白的化学位移进行了比较。在两种形式的BLA之间观察到的大变化主要局限于蛋白质的钙结合区域。这些数据表明，Na（+）通过在钙结合位点或其他地方屏蔽排斥性负电荷来稳定天然脱辅基状态，而不是通过在空的钙结合位点的特异性相互作用。与全蛋白形式相比，脱辅基形式中钙结合环和C螺旋中残基的氢交换保护作用降低。这表明在没有结合钙的情况下，蛋白质该区域的动态行为显著增加。实时NMR实验表明，与蛋白质从全蛋白形式转变为脱辅基形式相关的结构重排不涉及可检测到的部分未折叠中间体群体。相反，这种转变似乎涉及钙结合位点附近结构的局部重组，这些重组与蛋白质结构的内在波动相关联。
