Roos Matthias, Link Susanne, Balbach Jochen, Krushelnitsky Alexey, Saalwächter Kay
Institut für Physik, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale), Germany.
Institut für Physik, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale), Germany.
Biophys J. 2015 Jan 6;108(1):98-106. doi: 10.1016/j.bpj.2014.11.1858.
Knowledge about the global translational and rotational motion of proteins under crowded conditions is highly relevant for understanding the function of proteins in vivo. This holds in particular for human αB-crystallin, which is strongly crowded in vivo and inter alia responsible for preventing cataracts. Quantitative information on translational and rotational diffusion is not readily available, and we here demonstrate an approach that combines pulsed-field-gradient NMR for translational diffusion and proton T1ρ/T2 relaxation-time measurements for rotational diffusion, thus overcoming obstacles encountered in previous studies. The relaxation times measured at variable temperature provide a quantitative measure of the correlation function of protein tumbling, which cannot be approximated by a single exponential, because two components are needed for a minimal and adequate description of the data. We find that at high protein concentrations, rotational diffusion is decoupled from translational diffusion, the latter following the macroscopic viscosity change almost quantitatively, resembling the behavior of spherical colloids. Analysis of data reported in the literature shows that well-packed globular proteins follow a scaling relation between the hydrodynamic radius and the molar mass, Rh ∼ M(1/d), with a fractal dimension of d ∼ 2.5 rather than 3. Despite its oligomeric nature, Rh of αB-crystallin as derived from both NMR methods is found to be fully consistent with this relation.
了解在拥挤条件下蛋白质的整体平移和旋转运动对于理解蛋白质在体内的功能至关重要。这对于人类αB-晶状体蛋白尤其如此，它在体内高度拥挤，尤其负责预防白内障。关于平移和旋转扩散的定量信息并不容易获得，我们在此展示了一种方法，该方法结合了用于平移扩散的脉冲场梯度核磁共振和用于旋转扩散的质子T1ρ/T2弛豫时间测量，从而克服了先前研究中遇到的障碍。在可变温度下测量的弛豫时间提供了蛋白质翻滚相关函数的定量度量，由于需要两个分量来对数据进行最小且充分的描述，因此不能用单一指数来近似。我们发现，在高蛋白质浓度下，旋转扩散与平移扩散解耦，后者几乎定量地跟随宏观粘度变化，类似于球形胶体的行为。对文献中报道的数据的分析表明，紧密堆积的球状蛋白质遵循流体动力学半径与摩尔质量之间的标度关系，Rh ∼ M(1/d)，分形维数为d ∼ 2.5而非3。尽管αB-晶状体蛋白具有寡聚性质，但通过两种核磁共振方法得出的其流体动力学半径与该关系完全一致。
