Zhang M, Vogel H J
Department of Biological Sciences, University of Calgary, Alberta, Canada.
J Biol Chem. 1993 Oct 25;268(30):22420-8.
The 7 Lys residues in mammalian calmodulin (CaM) were reductively methylated with 13C-enriched formaldehyde and studied by (1H,13C)-heteronuclear multiple quantum coherence (HMQC) NMR. The apo- and Ca(2+)-forms of CaM, as well as a complex with a 22-residue peptide which comprises the CaM binding region of myosin light chain kinase were studied. The complete assignment of the two-dimensional NMR spectra was obtained by site-directed mutagenesis (Lys-->Gln) of all the Lys. The pKa values for the individual Lys could be determined by pH titration experiments. In Ca(2+)-CaM, the pKa values range from 9.29 (Lys-75) to 10.23 (Lys-77). The Lys in apo-CaM have higher pKa values than those in Ca(2+)-CaM. The binding of the myosin light chain kinase peptide gives rise to an increase of the pKa values of Lys-148 and Lys-75 by 0.5 and 0.8 pH units, respectively; this results from the relocation of their side chains to a completely solvent accessible state. The changes in the pKa values upon binding Ca2+ or the myosin light chain kinase peptide show a remarkable correlation with earlier reported chemical reactivity changes. Thus, our results indicate that pKa values, rather than structural and steric effects, play the dominant role in determining the reactivity of Lys side chains towards small electrophilic chemical modification reagents. The methodology used here could prove useful for the determination of individual pKa values in other proteins.
用富含13C的甲醛对哺乳动物钙调蛋白（CaM）中的7个赖氨酸残基进行还原甲基化，并通过（1H，13C）-异核多量子相干（HMQC）核磁共振进行研究。研究了CaM的脱辅基形式和Ca(2+)形式，以及与包含肌球蛋白轻链激酶CaM结合区域的22个残基肽的复合物。通过对所有赖氨酸进行定点诱变（赖氨酸→谷氨酰胺）获得二维核磁共振谱的完全归属。单个赖氨酸的pKa值可通过pH滴定实验确定。在Ca(2+)-CaM中，pKa值范围为9.29（赖氨酸-75）至10.23（赖氨酸-77）。脱辅基CaM中的赖氨酸比Ca(2+)-CaM中的赖氨酸具有更高的pKa值。肌球蛋白轻链激酶肽的结合分别使赖氨酸-148和赖氨酸-75的pKa值增加0.5和0.8个pH单位；这是由于它们的侧链重新定位到完全可被溶剂接触的状态。结合Ca2+或肌球蛋白轻链激酶肽时pKa值的变化与早期报道的化学反应性变化具有显著相关性。因此，我们的结果表明，pKa值而非结构和空间效应在决定赖氨酸侧链对小型亲电化学修饰试剂的反应性方面起主导作用。这里使用的方法可能对测定其他蛋白质中的单个pKa值有用。
