Kinetic study of phosphorylation-dependent complex formation between the kinase-inducible domain (KID) of CREB and the KIX domain of CBP on a quartz crystal microbalance
Kinetic study of phosphorylation-dependent complex formation between the kinase-inducible domain (KID) of CREB and the KIX domain of CBP on a quartz crystal microbalance
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DOI：
10.1002/chem.200400030
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发表时间：
2004-12-03
影响因子：
4.3
通讯作者：
Okahata, Y
中科院分区：
文献类型：
--
作者：
Matsuno, H;Furusawa, H;Okahata, Y
我们报告了水溶液中 27 MHz 石英晶体微天平 (QCM) 上肽-肽相互作用的定量分析。已知转录因子 CREB（环 AMP 响应元件结合蛋白）的 KID（激酶诱导结构域）可与共激活剂 CBP（CREB 结合蛋白）的 KIX 结构域相互作用，并通过 KID Ser-133 的磷酸化来促进。 KIX 结构域肽（86 个氨基酸）通过聚乙二醇间隔基固定在 QCM 金电极表面。通过 QCM 的频率降低（质量增加）来检测 KID 肽（46 个氨基酸）与 KIX 肽的结合。由于 KID 肽的 Ser-133 磷酸化，从 QCM 测量中获得的最大结合量 (Deltam(max)) 和关联常数 (K-a) 均增加。 KIX 肽对磷酸化 (pKID) 和未磷酸化 KID 肽的 K-a 值分别为 (93 2) X 10(3) 和 (5 1) X 10(3) m(-1)。这种差异是通过 pKID 的解离速率常数 (k(-1)) 比 KID 小 20 倍来解释的，而结合速率常数 (k(1)) 与磷酸化无关。
We report quantitative analysis of peptide-peptide interactions on a 27 MHz quartz crystal microbalance (QCM) in aqueous solution. The KID (kinase-inducible domain) of transcription factor CREB (cyclic AMP response element binding protein) is known to interact with the KIX domain of coactivator CBP (CREB binding protein), facilitated by phosphorylation at Ser-133 of the KID. The KIX domain peptide (86 aa) was immobilized on the QCM gold electrode surface by means of a poly(ethylene glycol) spacer. Binding of the KID peptide (46 aa) to the KIX peptide was detected by frequency decreases (mass increases) of the QCM. Both maximum binding amount (Deltam(max)) and association constants (K-a) obtained from the QCM measurements increased as a result of phosphorylation of Ser-133 of the KID peptide. The K-a values for KIX peptide to the phosphorylated (pKID) and unphosphorylated KID peptides were (93 2) X 10(3) and (5 1) X 10(3) m(-1), respectively This difference was explained by the dissociation rate constant (k(-1)) of the pKID being 20 times smaller than that of the KID, while association rate constants (k(1)) were independent of phosphorylation.