Structural characterization of two alternate conformations in a calbindin D₉k-based molecular switch.
Structural characterization of two alternate conformations in a calbindin D₉k-based molecular switch.
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DOI：
10.1021/bi102040g
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发表时间：
2011-06-28
期刊：
影响因子：
2.9
通讯作者：
Loh SN
中科院分区：
文献类型：
--
作者：
Stratton MM;McClendon S;Eliezer D;Loh SN
我们已经证明，钙结合蛋白D9 k可以通过复制蛋白质的C端一半（残基44-75）并将其附加到N端（产生残基44′-75′）而转化为钙敏感开关（钙结合蛋白-AFF）。这种再工程导致配体驱动的两个天然折叠之间的相互转换：野生型结构（N）和环状排列形式（N′）。据预测，N和N′之间的转换涉及44-75和44′-75′片段的交换，可能与它们各自的折叠和解折叠有关。在这里，我们提出了支持N和N′存在的直接结构证据。为了分离N′和N构象，我们在65位（E65 ′ Q突变体）或类似的65′位（E65′Q突变体）引入敲低的Ca 2+结合突变Glu → Gln。因此，E65 Q和E65′Q在钙的存在下，预计分别采用构象N′和N。虽然E65 Q和E65′Q的氨基酸序列仅在这两个位置不同，但核磁共振分配、化学位移和顺磁弛豫增强数据表明，它们在与钙结合时呈现不同的结构。这两种蛋白质都由折叠良好的结构域和无序区域组成。然而，在E65 Q中无序的片段（残基44-75）在E65′Q中折叠，并且在E65′Q中无序的区域（残基44′-75′）在E65 Q中结构化。结果表明，N <$N′构象变化是由一个相互排斥的折叠反应介导的，其中蛋白质的一个片段的折叠与另一个片段的去折叠偶联，反之亦然.
We have demonstrated that calbindin D9k can be converted into a calcium-sensing switch (calbindin-AFF) by duplicating the C-terminal half of the protein (residues 44–75) and appending it to the N-terminus (creating residues 44′–75′). This re-engineering results in a ligand-driven interconversion between two native folds: the wild-type structure (N) and a circularly permuted form (N′). The switch between N and N′ is predicted to involve exchange of the 44–75 and 44′–75′ segments, possibly linked to their respective folding and unfolding. Here we present direct structural evidence supporting the existence of N and N′. To isolate the N′ and N conformations, we introduced the knockdown Ca2+ binding mutation Glu → Gln at position 65 (E65Q mutant) or at the analogous position 65′ (E65′Q mutant). E65Q and E65′Q are therefore expected to adopt conformations N′ and N, respectively, in the presence of calcium. Though the amino acid sequences of E65Q and E65′Q differ at only these two positions, nuclear magnetic resonance resonance assignments, chemical shifts, and paramagnetic relaxation enhancement data reveal that they take on separate structures when bound to calcium. Both proteins are comprised of a well-folded domain and a disordered region. However, the segment that is disordered in E65Q (residues 44–75) is folded in E65′Q, and the region that is disordered in E65′Q (residues 44′–75′) is structured in E65Q. The results demonstrate that the N ⇆ N′ conformational change is mediated by a mutually exclusive folding reaction in which folding of one segment of the protein is coupled to unfolding of another segment, and vice versa.
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DOI：
10.1016/j.bbapap.2010.01.017
发表时间：
2010-06
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Uversky VN;Dunker AK
通讯作者：
Dunker AK
影响因子：
6.8
作者：
Dunker, A. Keith;Silman, Israel;Sussman, Joel L.
通讯作者：
Sussman, Joel L.
影响因子：
5.6
作者：
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通讯作者：
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DOI：
10.1002/prot.10080
发表时间：
2002-05-15
期刊：
PROTEINS-STRUCTURE FUNCTION AND GENETICS
影响因子：
--
作者：
Julenius, K;Robblee, J;Linse, S
通讯作者：
Linse, S
影响因子：
6.8
作者：
Eliezer, David
通讯作者：
Eliezer, David