Berggård T, Thulin E, Akerfeldt K S, Linse S
Physical Chemistry 2, Chemical Center, University of Lund, Sweden.
Protein Sci. 2000 Nov;9(11):2094-108. doi: 10.1110/ps.9.11.2094.
Calbindin D28k is a highly conserved Ca2+-binding protein abundant in brain and sensory neurons. The 261-residue protein contains six EF-hands packed into one globular domain. In this study, we have reconstituted calbindin D28k from two fragments containing three EF-hands each (residues 1-132 and 133-261, respectively), and from other combinations of small and large fragments. Complex formation is studied by ion-exchange and size-exclusion chromatography, electrophoresis, surface plasmon resonance, as well as circular dichroism (CD), fluorescence, and NMR spectroscopy. Similar chromatographic behavior to the native protein is observed for reconstituted complexes formed by mixing different sets of complementary fragments, produced by introducing a cut between EF-hands 1, 2, 3, or 4. The C-terminal half (residues 133-261) appears to have a lower intrinsic stability compared to the N-terminal half (residues 1-132). In the presence of Ca2+, NMR spectroscopy reveals a high degree of structural similarity between the intact protein and the protein reconstituted from the 1-132 and 133-261 fragments. The affinity between these two fragments is 2 x 10(7) M(-1), with association and dissociation rate constants of 2.7 x 10(4) M(-1) s(-1) and 1.4 x 10(-3) s(-1), respectively. The complex formed in the presence of Ca2+ is remarkably stable towards unfolding by urea and heat. Both the complex and intact protein display cold and heat denaturation, although residual alpha-helical structure is seen in the urea denatured state at high temperature. In the absence of Ca2+, the fragments do not recombine to yield a complex resembling the intact apo protein. Thus, calbindin D28k is an example of a protein that can only be reconstituted in the presence of bound ligand. The alpha-helical CD signal is increased by 26% after addition of Ca2+ to each half of the protein. This suggests that Ca2+-induced folding of the fragments is important for successful reconstitution of calbindin D28k.
钙结合蛋白D28k是一种高度保守的Ca2+结合蛋白，在脑和感觉神经元中大量存在。这种由261个氨基酸残基组成的蛋白质包含六个EF手结构，它们堆积成一个球状结构域。在本研究中，我们从两个分别包含三个EF手结构的片段（分别为残基1 - 132和133 - 261）以及其他大小片段的组合中重构了钙结合蛋白D28k。通过离子交换色谱、尺寸排阻色谱、电泳、表面等离子体共振以及圆二色性（CD）、荧光和核磁共振（NMR）光谱研究复合物的形成。对于通过混合不同组互补片段形成的重构复合物，观察到其色谱行为与天然蛋白相似，这些互补片段是通过在EF手结构1、2、3或4之间引入切割产生的。与N端一半（残基1 - 132）相比，C端一半（残基133 - 261）的内在稳定性似乎较低。在Ca2+存在下，核磁共振光谱显示完整蛋白与由1 - 132和133 - 261片段重构的蛋白之间具有高度的结构相似性。这两个片段之间的亲和力为2×10(7) M(-1)，缔合和解离速率常数分别为2.7×10(4) M(-1) s(-1)和1.4×10(-3) s(-1)。在Ca2+存在下形成的复合物对尿素和热诱导的解折叠具有显著的稳定性。复合物和完整蛋白都表现出冷变性和热变性，尽管在高温下尿素变性状态下仍可见残余的α-螺旋结构。在没有Ca2+的情况下，这些片段不会重新组合形成类似于完整脱辅基蛋白的复合物。因此，钙结合蛋白D28k是一种只有在结合配体存在时才能重构的蛋白质的例子。向蛋白的每一半添加Ca2+后，α-螺旋CD信号增加了26%。这表明Ca2+诱导的片段折叠对于钙结合蛋白D28k的成功重构很重要。
