O'Donnell Susan E, Newman Rhonda A, Witt Travis J, Hultman Rainbo, Froehlig John R, Christensen Adam P, Shea Madeline A
Department of Biochemistry, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.
Methods Enzymol. 2009;466:503-26. doi: 10.1016/S0076-6879(09)66021-3. Epub 2009 Nov 13.
Calmodulin (CaM) is a small (148 amino acid), ubiquitously expressed eukaryotic protein essential for Ca(2+) regulation and signaling. This highly acidic polypeptide (pI<4) has two homologous domains (N and C), each consisting of two EF-hand Ca(2+)-binding sites. Despite significant homology, the domains have intrinsic differences in their Ca(2+)-binding properties and separable roles in regulating physiological targets such as kinases and ion channels. In mammalian full-length CaM, sites III and IV in the C-domain bind Ca(2+) cooperatively with ~10-fold higher affinity than sites I and II in the N-domain. However, the difference is only twofold when CaM is severed at residue 75, indicating that anticooperative interactions occur in full-length CaM. The Ca(2+)-binding properties of sites I and II are regulated by several factors including the interplay of interdomain linker residues far from the binding sites. Our prior thermodynamic studies showed that these residues inhibit thermal denaturation and decrease calcium affinity. Based on high-resolution structures and NMR spectra, there appear to be interactions between charged residues in the sequence 75-80 and those near the amino terminus of CaM. To explore electrostatic contributions to interdomain interactions in CaM, KCl was used to perturb the Ca(2+)-binding affinity, thermal stability, and hydrodynamic size of a nested set of recombinant mammalian CaM (rCaM) fragments terminating at residues 75, 80, 85, or 90. Potassium chloride is known to decrease Ca(2+)-binding affinity of full-length CaM. It may act directly by competition with acidic side chains that chelate Ca(2+) in the binding sites, and indirectly elsewhere in the molecule by changing tertiary constraints and conformation. In all proteins studied, KCl decreased Ca(2+)-affinity, decreased Stokes radius, and increased thermal stability, but not monotonically. Crystallographic structures of Ca(2+)-saturated rCaM(1-75) (3B32.pdb) and rCaM(1-90) (3IFK.pdb) were determined, offering cautionary notes about the effect of packing interactions on flexible linkers. This chapter describes an array of methods for characterizing system-specific thermodynamic properties that in concert govern structure and function.
钙调蛋白（CaM）是一种小蛋白（148个氨基酸），在真核生物中普遍表达，对钙离子（Ca(2+)）的调节和信号传导至关重要。这种高度酸性的多肽（pI<4）有两个同源结构域（N和C），每个结构域由两个EF手型钙离子结合位点组成。尽管有显著的同源性，但这些结构域在钙离子结合特性以及调节激酶和离子通道等生理靶点方面具有不同的作用。在哺乳动物全长CaM中，C结构域中的位点III和IV协同结合钙离子，其亲和力比N结构域中的位点I和II高约10倍。然而，当CaM在第75位残基处切断时，差异仅为两倍，这表明在全长CaM中存在反协同相互作用。位点I和II的钙离子结合特性受多种因素调节，包括远离结合位点的结构域间连接残基的相互作用。我们之前的热力学研究表明，这些残基抑制热变性并降低钙亲和力。基于高分辨率结构和核磁共振光谱，在序列75 - 80中的带电残基与CaM氨基末端附近的残基之间似乎存在相互作用。为了探究静电作用对CaM结构域间相互作用的贡献，使用氯化钾来扰动一组以第75、80、85或90位残基终止的重组哺乳动物CaM（rCaM）片段的钙离子结合亲和力、热稳定性和流体力学尺寸。已知氯化钾会降低全长CaM的钙离子结合亲和力。它可能通过与结合位点中螯合钙离子的酸性侧链竞争而直接起作用，并且通过改变分子其他部位的三级结构限制和构象而间接起作用。在所有研究的蛋白质中，氯化钾降低了钙离子亲和力，减小了斯托克斯半径，并提高了热稳定性，但并非呈单调变化。确定了钙离子饱和的rCaM(1 - 75)（3B32.pdb）和rCaM(1 - 90)（3IFK.pdb）的晶体结构，这为堆积相互作用对柔性连接子的影响提供了警示。本章描述了一系列用于表征共同决定结构和功能的系统特异性热力学性质的方法。
