André Ingemar, Kesvatera Tõnu, Jönsson Bo, Akerfeldt Karin S, Linse Sara
Department of Biophysical Chemistry, Lund University, Chemical Center, SE-22100 Lund, Sweden.
Biophys J. 2004 Sep;87(3):1929-38. doi: 10.1529/biophysj.104.040998.
The complex between calmodulin and the calmodulin-binding portion of smMLCKp has been studied. Electrostatic interactions have been anticipated to be important in this system where a strongly negative protein binds a peptide with high positive charge. Electrostatic interactions were probed by varying the pH in the range from 4 to 11 and by charge deletions in CaM and smMLCKp. The change in net charge of CaM from approximately -5 at pH 4.5 to -15 at pH 7.5 leaves the binding constant virtually unchanged. The affinity was also unaffected by mutations in CaM and charge substitutions in the peptide. The insensitivity of the binding constant to pH may seem surprising, but it is a consequence of the high charge on both protein and peptide. At low pH it is further attenuated by a charge regulation mechanism. That is, the protein releases a number of protons when binding the positively charged peptide. We speculate that the role of electrostatic interactions is to discriminate against unbound proteins rather than to increase the affinity for any particular target protein.
已经对钙调蛋白与平滑肌肌球蛋白轻链激酶（smMLCKp）的钙调蛋白结合部分之间的复合物进行了研究。在这个系统中，一种带强负电荷的蛋白质与带高正电荷的肽结合，静电相互作用被认为很重要。通过在4至11的范围内改变pH值以及对钙调蛋白（CaM）和平滑肌肌球蛋白轻链激酶（smMLCKp）进行电荷缺失来探究静电相互作用。钙调蛋白的净电荷从pH 4.5时的约 -5变为pH 7.5时的 -15，但结合常数实际上保持不变。亲和力也不受钙调蛋白中的突变和肽中的电荷取代的影响。结合常数对pH不敏感可能看起来令人惊讶，但这是蛋白质和肽上高电荷的结果。在低pH时，它会因电荷调节机制而进一步减弱。也就是说，蛋白质在结合带正电荷的肽时会释放一些质子。我们推测静电相互作用的作用是区分未结合的蛋白质，而不是增加对任何特定靶蛋白的亲和力。
