A model for circular dichroism monitored dimerization and calcium binding in an EF-hand synthetic peptide
A model for circular dichroism monitored dimerization and calcium binding in an EF-hand synthetic peptide
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DOI：
10.1006/jtbi.1999.0947
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发表时间：
1999-07-21
影响因子：
2
通讯作者：
Reid, RE
中科院分区：
文献类型：
--
作者：
Franchini, PLA;Reid, RE
EF-手肽已显示结合钙并二聚化以形成完整的蛋白质结构域[Shaw，G.S.，Hedges，R.S. & Sykes，BD（1990年）。Science，249，280-283].当拟合单位点钙结合模型时，具有鲤鱼小清蛋白CD位点序列的合成33个残基EF-手肽显示表观钙解离常数增加7倍，而肽浓度降低8倍。该观察结果与EF-手二聚化一致。本文介绍了一种方法来确定二聚解离常数和钙解离常数的单体和二聚体形式的EF-手肽使用圆二色性技术。通过在钙饱和条件下监测222 nm处的负摩尔椭圆率随肽浓度的增加而增加，测定合成的小清蛋白CD位点的二聚解离常数为55.68 +/-10.76 μ M。使用二聚化常数，通过监测在添加增加量的钙时肽溶液的椭圆率的变化来确定该肽的单体和二聚体形式的钙解离常数。将该数据拟合到考虑到二聚化的数学模型，得到单体和二聚体形式的钙解离常数分别为421.3 +/- 21.56和47.06 +/- 6.72 μ M。(C)北京：科学出版社.
EF-hand peptides have been shown to bind calcium and dimerize to form an intact protein domain [Shaw, G.S., Hedges, R.S. & Sykes, B.D. (1990). Science, 249, 280-283]. A synthetic 33-residue EF-hand peptide with the sequence of carp parvalbumin CD site demonstrated a seven-fold increase in the apparent calcium dissociation constant with a eight-fold decrease in peptide concentration when fit to a single-site calcium-binding model. This observation is consistent with EF-hand dimerization. This paper describes a method to determine the dimerization dissociation constant and the calcium dissociation constants for both the monomer and dimer forms of this EF-hand peptide using circular dichroism techniques. By monitoring the increase in negative molar ellipticity at 222 nm with increasing peptide concentration under calcium-saturating conditions the dimerization dissociation constant for the synthetic parvalbumin CD site was determined to be 55.68 +/- 10.76 mu M. Using the dimerization constant, the calcium dissociation constants for both the monomer and dimer forms of this peptide were determined by monitoring the change in ellipticity of peptide solutions on addition of increasing amounts of calcium. A fit of this data to a mathematical model that takes into account dimerization results in calcium dissociation constants of 421.3 +/- 21.56 and 47.06 +/- 6.72 mu M for the monomer and dimer forms, respectively. (C) 1999 Academic Press.