RVCaB, a calcium-binding protein in radish vacuoles, is predominantly an unstructured protein with a polyproline type II helix
RVCaB, a calcium-binding protein in radish vacuoles, is predominantly an unstructured protein with a polyproline type II helix
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DOI：
10.1093/jb/mvm130
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发表时间：
2007-08-01
影响因子：
2.7
通讯作者：
Miyano, Masashi
中科院分区：
文献类型：
--
作者：
Ishijima, Jun;Nagasaki, Nahoko;Miyano, Masashi
萝卜液泡中存在一种独特的酸性钙结合蛋白RVCaB，富含谷氨酸和脯氨酸，缺乏芳香氨基酸残基，被认为参与液泡Ca2+的储存功能。在本研究中，我们重点研究了RVCaB的蛋白质物理化学性质，以了解其在结构和Ca2+结合功能方面的独特性。在差示扫描量热法上，除了从20℃加热到99℃时热容量逐渐过剩外，蛋白质折叠后没有表现出任何热变性的急剧转变。热处理后RVCaB的Ca2+结合能力保持不变。通过多种蛋白质结构分析程序判断，RVCaB的远ltv - CD光谱中未检测到α -螺旋或β -sheet。然而，进一步的CD光谱分析表明，RVCaB具有左旋脯氨酸11型（PPII）螺旋，已知为胶原链构象。测定与RVCaB结合的Ca2+数为21.6，通过等温滴定量热法测定Ca2+结合的360M(-1) Ka值。分析还表明，Ca2+与RVCaB的结合是一种熵驱动的现象。我们制备了RVCaB的色氨酸插入突变体（V136W和V202W），用荧光光谱检测Ca2+诱导的结构变化。分析表明，RVCaB在Ca2+结合时发生了小的结构重排，并且在每个突变体中，诱导的Trp残基136和202暴露于溶剂中。这些结果表明，除了扩展的PPII结构外，RVCaB没有确定的蛋白质折叠，并且其结构会因Ca2+结合或热处理而发生轻微变化。这些发现表明，RVCaB及其PPII螺旋的独特结构与其高容量和低亲和力的Ca2+结合特性密切相关。
A unique acidic calcium-binding protein RVCaB, rich in glutamic acid and proline and lacking aromatic amino-acid residues, exists in radish vacuoles, and is thought to be involved in the vacuole Ca2+-storage function. In the present study, we focused on the protein physicochemical properties of RVCaB to understand its uniqueness in terms of structure and Ca2+-binding function. On differential scanning calorimetry, the protein did not show any sharp transition of heat-denaturation of the folded protein except for a gradual excess of heat capacity when heated up to 99 degrees C from 20 degrees C. The Ca2+-binding ability of RVCaB was retained after heat treatment. No alpha-helix or beta-sheet was detected in the far-LTV CD spectra of RVCaB as judged by several computer programs for protein structure analysis. However, further analyses with CD spectroscopy suggest that RVCaB has a left-handed polyproline type 11 (PPII) helix, which is known to be in a collagen chain conformation. The number of Ca2+ bound to RVCaB was determined to be 21.6, and a 360M(-1) Ka value for Ca2+ binding was determined by isothermal titration calorimetry. The analysis also revealed that the binding of Ca2+ to RVCaB is an entropy-driven phenomenon. We prepared tryptophan-inserted mutants of RVCaB (V136W and V202W) to probe the Ca2+-induced structural change by fluorescent spectroscopy. The analysis suggests a small structural rearrangement of RVCaB upon Ca2+-binding and that the induced Trp residues at 136 and 202 are exposed to solvent in each mutant. These results suggest that RVCaB does not have a definitive protein fold except for the extended PPII structure and that its structure changes slightly by the binding of Ca2+ or heat treatment. These findings suggest that the unique structure of RVCaB with its PPII helices is closely related to its high-capacity and low-affinity Ca2+-binding properties.