Sorensen B R, Shea M A
Department of Biochemistry, University of Iowa College of Medicine, Iowa City 52242-1109, USA.
Biophys J. 1996 Dec;71(6):3407-20. doi: 10.1016/S0006-3495(96)79535-8.
Calmodulin (CaM) is an intracellular cooperative calcium-binding protein essential for activating many diverse target proteins. Biophysical studies of the calcium-induced conformational changes of CaM disagree on the structure of the linker between domains and possible orientations of the domains. Molecular dynamics studies have predicted that Ca4(2+)CaM is in equilibrium between an extended and compact conformation and that Arg74 and Arg90 are critical to the compaction process. In this study gel permeation chromatography was used to resolve calcium-induced changes in the hydrated shape of CaM at pH 7.4 and 5.6. Results showed that mutation of Arg 74 to Ala increases the R(s) as predicted; however, the average separation of domains in Ca4(2+)-CaM was larger than predicted by molecular dynamics. Mutation of Arg90 to Ala or Gly affected the dimensions of apo-CaM more than those of Ca4(2+)-CaM. Calcium binding to CaM and mutants (R74A-CaM, R90A-CaM, and R90G-CaM) lowered the Stokes radius (R(s)). Differences between R(s) values reported here and Rg values determined by small-angle x-ray scattering studies illustrate the importance of using multiple techniques to explore the solution properties of a flexible protein such as CaM.
钙调蛋白（CaM）是一种细胞内的协同钙结合蛋白，对于激活多种不同的靶蛋白至关重要。关于钙诱导的CaM构象变化的生物物理研究，在结构域之间的连接结构以及结构域的可能取向方面存在分歧。分子动力学研究预测，Ca4(2+)CaM在伸展构象和紧凑构象之间处于平衡状态，并且精氨酸74（Arg74）和精氨酸90（Arg90）对压缩过程至关重要。在本研究中，使用凝胶渗透色谱法来解析在pH 7.4和5.6条件下钙诱导的CaM水合形状的变化。结果表明，将Arg 74突变为丙氨酸（Ala）会如预测的那样增加斯托克斯半径（R(s)）；然而，Ca4(2+)-CaM中结构域的平均间距大于分子动力学预测的值。将Arg90突变为Ala或甘氨酸（Gly）对脱辅基CaM尺寸的影响大于对Ca4(2+)-CaM尺寸的影响。钙与CaM及其突变体（R74A-CaM、R90A-CaM和R90G-CaM）结合会降低斯托克斯半径（R(s)）。此处报道的R(s)值与通过小角X射线散射研究确定的Rg值之间的差异，说明了使用多种技术来探索像CaM这样的柔性蛋白质溶液性质的重要性。
