Rotational dynamics of calcium-free calmodulin studied by 15N-NMR relaxation measurements.
Rotational dynamics of calcium-free calmodulin studied by 15N-NMR relaxation measurements.
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DOI：
10.1111/j.1432-1033.1995.1014g.x
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发表时间：
1995-06
期刊：
影响因子：
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通讯作者：
Nico Tjandra;Hitoshi Kuboniwa;Hao Ren;Ad Bax
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文献类型：
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作者：
Nico Tjandra;Hitoshi Kuboniwa;Hao Ren;Ad Bax
无钙非洲爪蟾钙调蛋白的骨架运动的特征在于在51和61 MHz的15 N纵向弛豫时间（T1）的测量，并通过进行横向弛豫（T2），自旋锁定横向弛豫（T1 ρ），和15 N-[1H] heterogeneous NOE测量在61 MHz 15 N频率。虽然骨架酰胺氢交换实验表明N-末端结构域比钙调蛋白的C-末端半更稳定，但在所有八个α-螺旋和四个短β-链中的三个中发现了缓慢交换的骨架酰胺质子。这证实了无钙形式由稳定的二级结构组成，并且不采用“熔融球”类型的结构。然而，钙调蛋白的C-末端结构域在约350微秒的时间尺度上进行构象交换，这影响许多C-末端结构域残基。这导致15 N T2值相对于T1 rho显著缩短，而T1 rho和T2值在蛋白质的N-末端一半中具有相似的幅度。一个模型，其中的蛋白质的运动被假定为各向同性的蛋白质的旋转相关时间约为8纳秒，但定量不同意与磁场的依赖性的T1值，并没有解释不同的T2值发现不同的α-螺旋在N-末端域。这些后者的参数是兼容的一个灵活的哑铃模型，其中钙调蛋白的两个域中的每一个自由扩散在一个圆锥体的半角度约30度和时间常数约3纳秒，而蛋白质的整体旋转发生在一个慢得多的时间尺度约12纳秒。在螺旋C和D中的酰胺之间观察到的横向弛豫速率的差异表明，钙结合时螺旋间角的变化小于Herzberg等人Strynadka和James [Strynadka，N. C. J. & James，M. N. G. 05 The Dog of the Woman（1988）Genet. 3，1-17]。
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].