Seeholzer S H, Cohn M, Putkey J A, Means A R, Crespi H L
Proc Natl Acad Sci U S A. 1986 Jun;83(11):3634-8. doi: 10.1073/pnas.83.11.3634.
Completely deuterated calmodulin ([2H]CaM) has been prepared by expressing the chicken gene for CaM in Escherichia coli grown in 2H2O on a deuterated medium. The structural and dynamic properties of a 1:1 CaM/melittin (Mel) complex have been investigated by proton NMR. The spectrum of bound Mel is obtained directly from the spectrum of the [2H]CaM X Mel complex and is found to resemble strongly the spectrum of the helical species in methanol rather than that of the random coil species in water. The spectrum of bound CaM is obtained indirectly from the difference spectrum between [1H]CaM X Mel and [2H]CaM X Mel. Many changes are observed between free and bound CaM and they are distributed in both halves of the molecule, indicating that the binding of Mel affects the structure in both parts of the molecule. The rates of exchange of the amide protons of [2H]CaM with 2H2O were compared to those of [2H]CaM X Mel. The results showed that most, but not all, of the protons exchanged more slowly in the complex; after 40 hr, the residual peaks number 7 in CaM and greater than 20 in the complex. Again, changes in rates in CaM due to binding of Mel occurred in both halves of the molecule. The relative rates of amide proton exchange in CaM and its complex with Mel prove to be a sensitive criterion of differences in conformational stability and/or structure.
通过在以重水为原料的重水培养基中生长的大肠杆菌中表达鸡源钙调蛋白（CaM）基因，制备出了完全氘代的钙调蛋白（[2H]CaM）。利用质子核磁共振研究了1:1的CaM/蜂毒素（Mel）复合物的结构和动力学性质。结合态Mel的光谱直接从[2H]CaM×Mel复合物的光谱中获得，发现其与甲醇中螺旋态的光谱非常相似，而非与水中无规卷曲态的光谱相似。结合态CaM的光谱通过[1H]CaM×Mel和[2H]CaM×Mel之间的差谱间接获得。在游离态和结合态CaM之间观察到许多变化，且这些变化分布在分子的两半部分，这表明Mel的结合影响了分子两部分的结构。将[2H]CaM的酰胺质子与2H2O的交换速率与[2H]CaM×Mel的交换速率进行了比较。结果表明，复合物中大多数（但不是全部）质子的交换更慢；40小时后，CaM中残留峰为7个，复合物中大于20个。同样，由于Mel的结合导致CaM中交换速率的变化发生在分子的两半部分。CaM及其与Mel的复合物中酰胺质子交换的相对速率被证明是构象稳定性和/或结构差异的敏感指标。
