Mapping protein interfaces with a fluorogenic cross-linker and mass spectrometry: application to nebulin-calmodulin complexes.
Mapping protein interfaces with a fluorogenic cross-linker and mass spectrometry: application to nebulin-calmodulin complexes.
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DOI：
10.1021/bi010259
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发表时间：
2001-07
期刊：
影响因子：
2.9
通讯作者：
A. Sinz;Kuan Wang
中科院分区：
文献类型：
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作者：
A. Sinz;Kuan Wang
Nebulin是一种巨大的多功能蛋白质，被认为是骨骼肌肌节细丝上的长度调节蛋白标尺和钙/CaM介导的调节蛋白。为了定义星云蛋白和钙调素之间的分子界面，我们硫醇化赖氨酸的钙调素和ND 66，一个四模块克隆片段从星云蛋白的C-末端，与2-亚氨基硫杂环戊烷和交联的复合物与二溴异丙亚胺，烷基化硫醇对彼此之间约6 A，形成荧光加合物。这种两阶段交联主要产生ND 66和CaM的1：1复合物，具有有限程度的分子内交联。凝胶内胰凝乳蛋白酶消化的二溴丙烷交联的复合物产生的肽，首先通过荧光检测的HPLC筛选，然后评分与质谱交联。通过ESI-MS/MS实验进一步鉴定和确认了几个分子间和分子内位点，定义了分子界面和蛋白质折叠模式。特别是，在ND 66中氨基酸83-99（YKENMGKGTPLPVTPEM）区域内的序列的5个分子间交联产物和钙调素的几个序列表明，星云蛋白-钙调素界面接近，并可能与星云蛋白-肌动蛋白界面重叠。这种接近表明钙调素和肌动蛋白之间的竞争，这星云接口。氨基酸13-16（KEAF）和13-18（KEAFSL）与氨基酸145-148（MTAK）和146-148（TAK）在CaM中的分子内交联表明两个叶跨中心螺旋的相互作用。ND 66中氨基酸1-6（MKTPEM）与氨基酸114-129（YKENVGKATATPVTPE）和115-129（KENVGKATATPVTPE）的交联暗示了溶液中非连续星云蛋白模块的缔合。
Nebulin is a giant multifunctional protein that is thought to serve as both a length-regulating protein ruler and calcium/CaM-mediated regulatory protein on the thin filaments of the skeletal muscle sarcomere. To define molecular interfaces between nebulin and CaM, we thiolated lysines of CaM and ND66, a four-module cloned fragment from the C-terminus of nebulin, with 2-iminothiolane and cross-linked the complex with dibromobimane, which alkylates thiol pairs within approximately 6 A of each other to form a fluorescent adduct. Such a two-stage cross-linking generated mainly 1:1 complexes of ND66 and CaM, with a limited extent of intramolecular cross-linking. In-gel chymotryptic digestion of the dibromobimane-cross-linked complexes yielded peptides that were first screened by HPLC with fluorescence detection and then scored for cross-linking with mass spectrometry. Several inter- and intramolecular sites were identified and confirmed further by ESI-MS/MS experiments, defining molecular interfaces and patterns of protein folding. In particular, five intermolecular cross-linking products of sequences within the region of amino acids 83-99 (YKENMGKGTPLPVTPEM) in ND66 and several sequences of CaM indicate that the nebulin-CaM interface is close to, and may overlap with, the nebulin-actin interface. This proximity suggests a potential competition between CaM and actin for this nebulin interface. Intramolecular cross-linking of amino acids 13-16 (KEAF) and 13-18 (KEAFSL) with amino acids 145-148 (MTAK) and 146-148 (TAK) in CaM suggests the interaction of two lobes across the central helix. The cross-linking of amino acids 1-6 (MKTPEM) with amino acids 114-129 (YKENVGKATATPVTPE) and 115-129 (KENVGKATATPVTPE) in ND66 hints at an association of noncontiguous nebulin modules in solution.