EUROPIUM(III) LUMINESCENCE AND TYROSINE TO TERBIUM(III) ENERGY-TRANSFER STUDIES OF INVERTEBRATE (OCTOPUS) CALMODULIN
EUROPIUM(III) LUMINESCENCE AND TYROSINE TO TERBIUM(III) ENERGY-TRANSFER STUDIES OF INVERTEBRATE (OCTOPUS) CALMODULIN
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DOI：
10.1021/bi00146a002
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发表时间：
1992-08-11
期刊：
影响因子：
2.9
通讯作者：
ZAUHAR, RJ
中科院分区：
文献类型：
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作者：
BRUNO, J;HORROCKS, WD;ZAUHAR, RJ
通过激光诱导Eu3+和Tb3+发光研究了脊椎动物(牛睾丸)和无脊椎动物(章鱼)钙调素氨基酸序列的微小差异对金属离子结合的影响。与金属离子配位的残基上的氨基酸取代不会使与章鱼钙调蛋白结合的Eu3+离子的F-7(0)->；5D0激发光谱相对于牛睾丸钙调蛋白产生任何可检测到的变化；在D2O溶液中仅观察到激发态寿命值的微小差异。用发光法测定了Eu3+(1.0+/-0.2-mU-M)和Tb3+(5+/-1-mU-M)与章鱼钙调蛋白弱稀土结合部位(从氨基末端编号)的解离常数。这两个值都与先前报道的牛睾丸钙调蛋白[MulQueen，P.M.，Tingey，J.M.，&Horrocks，W.D.，Jr.(1985年)生物化学24,6639-66451。测得稀土元素紧密结合部位(I和II)上Eu~(3+)的离解常数章鱼钙调素为6+/-2 nM，牛睾丸钙调素为12+/-2 nM。在含有Eu~(3+)供体和Nd~(3+)受体离子的章鱼钙调蛋白D2O溶液中，用Forster能量转移方法测定了第一和第二位点(12.4+/-0.5埃)和第三位点和第四位点(11.7+/-0.8埃)之间的距离。综合计算了章鱼钙调蛋白第III位和第IV位Tyr138和Tb3+离子之间非辐射能量转移的Forster理论参数，包括取向因子kappa-2的动力学模拟。这一理论被发现定量地解释了从观察到的敏化Tb3+发射评估的观察到的能量转移效率。
The effects of minor differences in the amino acid sequences between a vertebrate (bovine testes) and an invertebrate (octopus) calmodulin on metal ion binding were investigated via laser-induced Eu3+ and Tb3+ luminescence. Amino acid substitutions at residues which are coordinated to the metal ion do not produce any detectable changes in the F-7(0)-->5D0 excitation spectrum of the Eu3+ ion bound to octopus calmodulin relative to bovine testes calmodulin; only minor differences in the excited-state lifetime values in D2O solution are observed. The dissociation constants for Eu3+ (1.0 +/- 0.2-mu-M) and Tb3+ (5 +/- 1-mu-M) from the weak lanthanide binding sites (III and IV, numbered from the amino terminus) of octopus calmodulin were measured using luminescence techniques. Both values agree well with those reported previously for bovine testes calmodulin [Mulqueen, P. M., Tingey, J. M., & Horrocks, W. D., Jr. (1985) Biochemistry 24, 6639-66451. The measured dissociation constant of Eu3+ bound in the tight lanthanide binding sites (I and II) is 6 +/- 2 nM for octopus calmodulin and 12 +/- 2 nM for bovine testes calmodulin. The distances between sites I and II (12.4 +/- 0.5 angstrom) and sites III and IV (11.7 +/- 0.8 angstrom) were determined from Forster-type energy transfer in D2O Solutions of octopus calmodulin containing bound Eu3+ donor and Nd3+ acceptor ions. Forster theory parameters for nonradiative energy transfer between Tyr138 and Tb3+ ions bound at sites III and IV of octopus calmodulin were comprehensively evaluated, including a dynamics simulation of the orientation factor kappa-2. This theory is found to account quantitatively for the observed energy-transfer efficiency as evaluated from the observed sensitized Tb3+ emission.