Martin S R, Bayley P M, Brown S E, Porumb T, Zhang M, Ikura M
Division of Physical Biochemistry, National Institute for Medical Research, London, UK.
Biochemistry. 1996 Mar 19;35(11):3508-17. doi: 10.1021/bi952522a.
We describe the properties of a hybrid protein comprising the full length of the Xenopus laevis calmodulin sequence, followed by a pentapeptide linker (GGGGS), and residues 3-26 of M13, the calmodulin binding region of skeletal muscle myosin light chain kinase. The properties of the hybrid protein are compared with those of the complex formed between Drosophila calmodulin and a peptide corresponding to residues 1-18 of the M13 sequence. The addition of calcium to the hybrid protein produces pronounced changes in the near- and far-UV CD spectra, in the fluorescence emission spectrum of the single tryptophan residue at position 4 in the M13 sequence, and in the accessibility of this tryptophan residue to acrylamide quenching. These changes are consistent with the tryptophan residue being immobilized in a hydrophobic environment and with the hybrid protein adopting a more alpha-helical structure when calcium is bound. The increased alpha-helicity derives from changes in both the calmodulin and peptide regions of the hybrid protein. Changes in the circular dichroism and fluorescence properties of the hybrid protein as a function of the calcium to hybrid protein ratio are consistent with the fact that these changes parallel the cooperative binding of all four calcium ions. The hybrid protein shows greatly increased affinity (>250-fold) for calcium compared with calmodulin itself. Macroscopic calcium binding constants (K(1)-K(4)) were determined from calcium titrations performed in the presence of the calcium chelator Quin 2. Values for log(K(1)K(2)) and log(K(3)K(4)) were determined to be 15.4 +/- 0.2 and 15.59 +/- 0.22 (20 degrees C). The corresponding values for Drosophila calmodulin alone are 11.65 +/- 0.15 and 9.66 +/- 0.25. Consistent with this increased affinity for calcium stopped-flow kinetic studies suggest that the dissociation rate for the N-terminal calcium ions is reduced to at least 0.77 s(-1), compared with approximately 700 s(-1) for Drosophila calmodulin in the absence of peptide. This hybrid protein illustrates the principle whereby the binding of a peptide sequence covalently attached to calmodulin can enhance the average calcium affinity by more than 2 orders of magnitude. Conversely, the target sequence in the hybrid protein undergoes a calcium-induced conformational change to bind to the calmodulin in a conformation very similar to that of the corresponding dissociable target sequence binding to calmodulin, but with a greatly enhanced affinity due to its physical proximity to the binding site. This avoidance of the energetic penalty of dissociation may be a key contributory factor in determining the high affinity and specificity of the complex multiple interactions involved in recognition of biological targets by calmodulin.
我们描述了一种杂交蛋白的特性，该蛋白包含非洲爪蟾钙调蛋白序列的全长，其后是一个五肽接头（GGGGS），以及骨骼肌肌球蛋白轻链激酶的钙调蛋白结合区域M13的第3至26位残基。将该杂交蛋白的特性与果蝇钙调蛋白和对应于M13序列第1至18位残基的肽形成的复合物的特性进行了比较。向杂交蛋白中添加钙会使近紫外和远紫外圆二色光谱、M13序列中第4位单个色氨酸残基的荧光发射光谱以及该色氨酸残基对丙烯酰胺淬灭的可及性发生显著变化。这些变化与色氨酸残基固定在疏水环境中以及钙结合时杂交蛋白采用更具α螺旋结构一致。α螺旋度的增加源于杂交蛋白的钙调蛋白和肽区域的变化。杂交蛋白的圆二色性和荧光特性随钙与杂交蛋白比例的变化与这些变化与所有四个钙离子的协同结合平行这一事实一致。与钙调蛋白本身相比，杂交蛋白对钙的亲和力大大增加（>250倍）。通过在钙螯合剂喹啉2存在下进行的钙滴定测定宏观钙结合常数（K(1)-K(4)）。log(K(1)K(2))和log(K(3)K(4))的值分别确定为15.4±0.2和15.59±0.22（20℃）。单独的果蝇钙调蛋白的相应值为11.65±0.15和9.66±0.25。与这种对钙的亲和力增加一致，停流动力学研究表明，与不存在肽时果蝇钙调蛋白的约700 s(-1)相比，N端钙离子的解离速率降低至至少0.77 s(-1)。这种杂交蛋白说明了这样一个原理，即与钙调蛋白共价连接的肽序列的结合可使平均钙亲和力提高超过两个数量级。相反，杂交蛋白中的靶序列会发生钙诱导的构象变化，以与钙调蛋白结合，其构象与相应的可解离靶序列与钙调蛋白结合的构象非常相似，但由于其与结合位点的物理接近性而具有大大增强的亲和力。避免解离的能量代价可能是决定钙调蛋白识别生物靶标所涉及的复杂多重相互作用的高亲和力和特异性的关键因素。
