Residue-specific pKa determination of lysine and arginine side chains by indirect 15N and 13C NMR spectroscopy:: Application to apo calmodulin
Residue-specific pKa determination of lysine and arginine side chains by indirect 15N and 13C NMR spectroscopy:: Application to apo calmodulin
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DOI：
10.1021/ja0721824
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发表时间：
2007-12-26
影响因子：
15
通讯作者：
Mulder, Frans A. A.
中科院分区：
文献类型：
--
作者：
Andre, Ingemar;Linse, Sara;Mulder, Frans A. A.
蛋白质中的静电相互作用可以通过测定残基特异性酸性常数来进行实验研究。本文介绍了三重共振NMR技术用于直接测定蛋白质中赖氨酸和精氨酸侧链质子化状态。该实验基于在整个pH和温度范围内检测不可交换质子，因此非常适合于单个氨基酸侧链的pK（a）测定。实验遵循侧链N-15（zeta）（赖氨酸）和N-15（zeta）或C-13（zeta）（精氨酸）化学位移，其变化是由于质子化（去质子化）后杂电子分布的相当大的变化。由于杂原子化学位移被氨基酸侧链本身的电荷状态所淹没，这些方法在准确性、灵敏度和选择性方面取代了基于H-1的NMR。此外，N-15（zeta）和N-15（zeta）核可用于探测局部静电环境的变化。描述了对三种蛋白质的应用：载脂蛋白钙调蛋白、钙结合蛋白D-9 k和FKBP 12。对于载脂蛋白钙调蛋白，由于蛋白质表面的高净负电荷，赖氨酸侧链的残基特异性pK（a）值确定为介于10.7和11.2之间。理想的双态滴定行为观察到的所有赖氨酸指示的碱性残基之间的显着的直接电荷相互作用的情况下。这些结果进行了比较与早期的研究的基础上化学修饰。
Electrostatic interactions in proteins can be probed experimentally through determination of residue-specific acidity constants, We describe here triple-resonance NMR techniques for direct determination of lysine and arginine side-chain protonation states in proteins. The experiments are based on detection of nonexchangeable protons over the full range of pH and temperature and therefore are well suited for pK(a) determination of individual amino acid side chains. The experiments follow the side-chain N-15(zeta) (lysine) and N-15(epsilon) or C-13(zeta) (arginine) chemical shift, which changes due to sizable changes in the heteronuclear electron distribution upon (de)protonation. Since heteronuclear chemical shifts are overwhelmed by the charge state of the amino acid side chain itself, these methods supersede H-1-based NMR in terms of accuracy, sensitivity, and selectivity. Moreover, the N-15(zeta) and N-15(epsilon) nuclei may be used to probe changes in the local electrostatic environment. Applications to three proteins are described: apo calmodulin, calbindin D-9k, and FKBP12. For apo calmodulin, residue-specific pK(a) values of lysine side chains were determined to fall between 10.7 and 11.2 as a result of the high net negative charge on the protein surface. Ideal two-state titration behavior observed for all lysines indicates the absence of significant direct charge interactions between the basic residues. These results are compared with earlier studies based on chemical modification.