Pauls T L, Durussel I, Berchtold M W, Cox J A
Institute of Veterinary Biochemistry, University of Zürich-Irchel, Switzerland.
Biochemistry. 1994 Aug 30;33(34):10393-400. doi: 10.1021/bi00200a021.
Previously a rat parvalbumin mutant protein PVF102W was constructed with a reporter Trp at position 102 in the middle of the hydrophobic center [Pauls, T. L., et al. (1993) J. Biol. Chem. 268, 20897-20903]. In the present study three new parvalbumin mutant proteins, derived from PVF102W and containing alterations at positions essential for Ca2+ binding in either one of the two Ca(2+)-binding sites (PV-CD and PV-EF) or in both (PV-CD/-EF), were expressed and purified. With the flow dialysis method it was established that both PV-CD and PV-EF bind 1 Ca2+ with affinity constants KCa of 1.1 x 10(7) and 3.2 x 10(6) M-1, respectively. Mg2+ binding, monitored by equilibrium gel filtration in the absence of Ca2+, showed that both mutants bind 1 Mg2+ with KMg = 8 10(4) for PV-CD and 3 x 10(3) M-1 for PV-EF. Compared to the parameters of the parent mutant PVF102W (two sites with equal affinities of 2.7 x 10(7) and 3 x 10(4) M-1 for Ca2+ and Mg2+, respectively), these data indicate that inactivation of the EF site, much more than of the CD site, impairs divalent cation binding. The binding of Ca2+ and Mg2+ is mutually exclusive, indicative of a Ca2+/Mg2+ mixed site. However, as for PVF102W, the KMg values obtained from the competition equation are approximately 40-fold lower than the affinities measured by direct binding. PV-CD/-EF binds neither Ca2+ nor Mg2+. Trp fluorimetry revealed that in the three mutant PVs the residue Trp-102 is deeply buried in the hydrophobic core.(ABSTRACT TRUNCATED AT 250 WORDS)
之前构建了一种大鼠小清蛋白突变蛋白PVF102W，其在疏水中心中部的第102位带有报告色氨酸[保罗斯，T. L.等人（1993年）《生物化学杂志》268卷，20897 - 20903页]。在本研究中，表达并纯化了三种新的小清蛋白突变蛋白，它们源自PVF102W，且在两个钙结合位点之一（PV - CD和PV - EF）或两者（PV - CD/-EF）中对钙结合至关重要的位置发生了改变。通过流动透析法确定，PV - CD和PV - EF均结合1个Ca²⁺，其亲和常数KCa分别为1.1×10⁷和3.2×10⁶ M⁻¹。在无Ca²⁺的情况下通过平衡凝胶过滤监测Mg²⁺结合，结果表明两个突变体均结合1个Mg²⁺，PV - CD的KMg = 8×10⁴，PV - EF的KMg = 3×10³ M⁻¹。与亲本突变体PVF102W的参数（两个位点对Ca²⁺和Mg²⁺的亲和力分别为2.7×10⁷和3×10⁴ M⁻¹）相比，这些数据表明EF位点的失活比CD位点的失活更严重地损害二价阳离子结合。Ca²⁺和Mg²⁺的结合相互排斥，表明是Ca²⁺/Mg²⁺混合位点。然而，与PVF102W一样，从竞争方程获得的KMg值比通过直接结合测量的亲和力低约40倍。PV - CD/-EF既不结合Ca²⁺也不结合Mg²⁺。色氨酸荧光法显示，在这三种突变型小清蛋白中，色氨酸残基Trp - 102深埋于疏水核心中。（摘要截短于250字）
