Tjandra N, Kuboniwa H, Ren H, Bax A
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520, USA.
Eur J Biochem. 1995 Jun 15;230(3):1014-24. doi: 10.1111/j.1432-1033.1995.tb20650.x.
The backbone motions of calcium-free Xenopus calmodulin have been characterized by measurements of the 15N longitudinal relaxation times (T1) at 51 and 61 MHz, and by conducting transverse relaxation (T2), spin-locked transverse relaxation (T1 rho), and 15N-[1H] heteronuclear NOE measurements at 61 MHz 15N frequency. Although backbone amide hydrogen exchange experiments indicate that the N-terminal domain is more stable than calmodulin's C-terminal half, slowly exchanging backbone amide protons are found in all eight alpha-helices and in three of the four short beta-strands. This confirms that the calcium-free form consists of stable secondary structure and does not adopt a 'molten globule' type of structure. However, the C-terminal domain of calmodulin is subject to conformational exchange on a time scale of about 350 microseconds, which affects many of the C-terminal domain residues. This results in significant shortening of the 15N T2 values relative to T1 rho, whereas the T1 rho and T2 values are of similar magnitude in the N-terminal half of the protein. A model in which the motion of the protein is assumed to be isotropic suggests a rotational correlation time for the protein of about 8 ns but quantitatively does not agree with the magnetic field dependence of the T1 values and does not explain the different T2 values found for different alpha-helices in the N-terminal domain. These latter parameters are compatible with a flexible dumb-bell model in which each of calmodulin's two domains freely diffuse in a cone with a semi-angle of about 30 degrees and a time constant of about 3 ns, whereas the overall rotation of the protein occurs on a much slower time scale of about 12 ns. The difference in the transverse relaxation rates observed between the amides in helices C and D suggests that the change in interhelical angle upon calcium binding is less than predicted by Herzberg et al. Strynadka and James [Strynadka, N. C. J. & James, M. N. G. (1988) Proteins Struct. Funct. Genet. 3, 1-17].
通过在51和61兆赫兹下测量15N纵向弛豫时间（T1），以及在61兆赫兹15N频率下进行横向弛豫（T2）、自旋锁定横向弛豫（T1ρ）和15N-[1H]异核NOE测量，对无钙非洲爪蟾钙调蛋白的主链运动进行了表征。尽管主链酰胺氢交换实验表明N端结构域比钙调蛋白的C端结构域更稳定，但在所有八个α螺旋和四个短β链中的三个中都发现了缓慢交换的主链酰胺质子。这证实了无钙形式由稳定的二级结构组成，不采用“熔球”类型的结构。然而，钙调蛋白的C端结构域在约350微秒的时间尺度上经历构象交换，这影响了许多C端结构域残基。这导致相对于T1ρ，15N T2值显著缩短，而在蛋白质的N端结构域中，T1ρ和T2值大小相似。一个假设蛋白质运动为各向同性的模型表明，蛋白质的旋转相关时间约为8纳秒，但在定量上与T1值的磁场依赖性不一致，也无法解释在N端结构域中不同α螺旋发现的不同T2值。后两个参数与一个灵活的哑铃模型兼容，在该模型中，钙调蛋白的两个结构域各自在一个半角约为30度、时间常数约为3纳秒的圆锥中自由扩散，而蛋白质的整体旋转发生在约12纳秒的慢得多的时间尺度上。在螺旋C和D中的酰胺之间观察到的横向弛豫率差异表明，钙结合时螺旋间角度的变化小于Herzberg等人、Strynadka和James [Strynadka, N. C. J. & James, M. N. G. (1988) Proteins Struct. Funct. Genet. 3, 1 - 17]所预测的值。
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