A CALMODULIN-TARGET PEPTIDE HYBRID MOLECULE WITH UNIQUE CALCIUM-BINDING PROPERTIES
A CALMODULIN-TARGET PEPTIDE HYBRID MOLECULE WITH UNIQUE CALCIUM-BINDING PROPERTIES
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DOI：
10.1093/protein/7.1.109
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发表时间：
1994-01-01
期刊：
影响因子：
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通讯作者：
IKURA, M
中科院分区：
文献类型：
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作者：
PORUMB, T;YAU, P;IKURA, M
本文描述了一种杂交蛋白的生产和特性，该杂交蛋白包括非洲爪蟾钙调素（CaM）序列的全长，随后通过甘氨酰甘氨酸接头，由肌球蛋白轻链激酶（M13）的26个残基的CaM结合区。这种杂合分子似乎具有高热稳定性（在Ca 2+存在下T（m）> 75 ℃）以及不寻常的Ca 2+结合特性：（i）宽范围的双相Ca 2+结合响应（延伸超过pCa 4.8-7.4）和（ii）高表观结合常数（pCa 50%= 6.3，比野生型CaM增加10倍）。NMR和CD数据表明，CaM-M13杂合分子以两种主要构象之间约1：1的比例平衡存在，其中一种类似于CaM-M13复合物的紧凑球形结构[M.Ikura，G. M. Clore，A. M. Gronenborn，G.Zhu，C. B. Klee和A.Bax（1992）Science，256，632 - 638]，另一种类似于野生型CaM的哑铃状结构[Y. S. Babu，C. E. Bugg和W. J. Cook（1988）J. Mol. Biol.生物学：204，191-2041。双相Ca 2+结合曲线可以使用具有显著不同解离常数（2 x 10（-6）M和8 x 10（-8）M）的两条Hill结合曲线的线性组合来解释，这可以归因于平衡时的两种构象。本研究开辟了一条途径，工程蛋白具有较高的钙离子结合亲和力使用已知的钙调素结构作为模板。
This paper describes the production and properties of a hybrid protein comprising the full length of the Xenopus laevis calmodulin (CaM) sequence, followed, through a glycylglycine linker, by the 26-residue CaM-binding region of myosin lightchain kinase (M13). This hybrid molecule appears to have high thermal stability (T(m) > 75-degrees-C in the presence of Ca2+) as well as unusual Ca2+-binding properties: (i) a wide-range biphasic Ca2+-binding response (extending over pCa 4.8-7.4) and (ii) a high apparent binding constant (pCa50% = 6.3, a 10-fold increase from that of wild-type CaM). NMR and CD data indicate that the CaM - M13 hybrid molecule exists in equilibrium in an approximate 1:1 ratio between two major conformations, one of which is similar to the compact globular structure of the CaM-M13 complex [M.Ikura, G.M.Clore, A.M.Gronenborn, G.Zhu, C.B.Klee and A.Bax (1992) Science, 256, 632 - 638] and the other to the dumb-bell-like structure of the wild type CaM [Y.S.Babu, C.E.Bugg and W.J.Cook (1988) J. Mol. Biol., 204, 191-2041. The biphasic Ca2+-binding curve can be interpreted using a linear combination of two Hill binding curves with significantly different dissociation constants (2 x 10(-6) M and 8 x 10(-8) M), which can be attributed to the two conformations in equilibrium. The present study has opened an avenue to engineer proteins with higher Ca2+-binding affinities using the known CaM structures as a template.