Coupling of ligand binding and dimerization of helix-loop-helix peptides:: Spectroscopic and sedimentation analyses of calbindin D9k EF-hands
Coupling of ligand binding and dimerization of helix-loop-helix peptides:: Spectroscopic and sedimentation analyses of calbindin D9k EF-hands
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DOI：
10.1002/prot.10080
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发表时间：
2002-05-15
期刊：
影响因子：
--
通讯作者：
Linse, S
中科院分区：
文献类型：
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作者：
Julenius, K;Robblee, J;Linse, S
分离的Ca2+结合EF-hand肽具有二聚化的倾向。本研究试图解释Ca2+结合和肽结合的耦合平衡，两个ef手具有显著不同的环序列和净电荷。我们研究了calbindin D-9k的两个单独的EF-hand片段。用CD和荧光光谱法监测了不同肽浓度下的一系列Ca2+滴定。所有数据同时拟合到所有可能的平衡中间体的完整模型和在没有Ca2+的情况下不包括二聚化的简化模型。分析性超离心表明，根据溶液条件，肽可能以单体或二聚体的形式出现。我们的研究结果显示，两只ef手的行为截然不同。含有n端EF-hand的片段在Ca2+结合状态下表现出强烈的二聚化倾向。平均Ca2+亲和力比完整蛋白低3.5个数量级。我们观察到Ca2+结合在从无Ca2+单体到满载二聚体的整个过程中具有明显的协同性，表明无Ca2+ EF-hand在二聚化时折叠成Ca2+结合的EF-hand，从而向第二个Ca2+离子提供预形成的结合位点。c端EF-hand表现出较小的二聚化倾向，这可能与其较大的净负电荷有关。尽管二聚化行为不同，但两种EF-hand片段的Ca2+亲和力相似，在IgK = 4.6-5.3范围内。(C) 2002 Wiley-Liss, Inc。
Isolated Ca2+-binding EF-hand peptides have a tendency to dimerize. This study is an attempt to account for the coupled equilibria of Ca2+-binding and peptide association for two EF-hands with strikingly different loop sequence and net charge. We have studied each of the two separate EF-hand fragments from calbindin D-9k. A series of Ca2+-titrations at different peptide concentrations were monitored by CD and fluorescence spectroscopy. All data were fitted simultaneously to both a complete model of all possible equilibrium intermediates and a reduced model not including dimerization in the absence of Ca2+. Analytical ultracentrifugation shows that the peptides may occur as monomers or dimers depending on the solution conditions. Our results show strikingly different behavior for the two EF-hands. The fragment containing the N-terminal EF-hand shows a strong tendency to dimerize in the Ca2+-bound state. The average Ca2+-affinity is 3.5 orders of magnitude lower than for the intact protein. We observe a large apparent cooperativity of Ca2+ binding for the overall process from Ca2+-free monomer to fully loaded dimer, showing that a Ca2+-free EF-hand folds upon dimerization to a Ca2+-bound EF-hand, thereby presenting a preformed binding site to the second Ca2+-ion. The C-terminal EF-hand shows a much smaller tendency to dimerize, which may be related to its larger net negative charge. In spite of the differences in dimerization behavior, the Ca2+ affinities of both EF-hand fragments are similar and in the range IgK = 4.6-5.3. (C) 2002 Wiley-Liss, Inc.