Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, USA.
Department of Chemistry, University of Connecticut, Storrs, Connecticut, USA.
Protein Sci. 2022 May;31(5):e4321. doi: 10.1002/pro.4321.
Hydrodynamic radii (R -values) calculated from diffusion coefficients measured by pulse-field-gradient nuclear magnetic resonance are compared for folded and unfolded proteins. For native globular proteins, the R -values increase as a power of 0.35 with molecular size, close to the scaling factor of 0.33 predicted from polymer theory. Unfolded proteins were studied under four sets of conditions: in the absence of denaturants, in the presence of 6 M urea, in 95% dimethyl sulfoxide (DMSO), and in 40% hexafluoroisopropanol (HFIP). Scaling factors under all four unfolding conditions are similar (0.49-0.53) approaching the theoretical value of 0.60 for a fully unfolded random coil. Persistence lengths are also similar, except smaller in 95% DMSO, suggesting that the polypeptides are more disordered on a local scale with this solvent. Three of the proteins in our unfolded set have an asymmetric sequence-distribution of charged residues. While these proteins behave normally in water and 6 M urea, they give atypically low R -values in 40% HFIP and 95% DMSO suggesting they are forming electrostatic hairpins, favored by their asymmetric sequence charge distribution and the low dielectric constants of DMSO and HFIP. While diffusion-ordered NMR spectroscopy can separate small molecules, we show a number of factors combine to make protein-sized molecules much more difficult to resolve in mixtures. Finally, we look at the temperature dependence of apparent diffusion coefficients. Small molecules show a linear temperature response, while large proteins show abnormally large apparent diffusion coefficients at high temperatures due to convection, suggesting diffusion reference standards are only useful near 25°C.
利用脉冲场梯度核磁共振测量扩散系数计算得到的水动力半径（R 值），比较了折叠和未折叠蛋白质。对于天然球状蛋白质，R 值随分子大小呈 0.35 的幂次增加，接近聚合物理论预测的 0.33 的比例因子。在四种条件下研究了展开的蛋白质：在没有变性剂的情况下、存在 6 M 脲的情况下、在 95%二甲基亚砜（DMSO）中、在 40%六氟异丙醇（HFIP）中。在所有四种展开条件下，比例因子相似（0.49-0.53），接近完全展开的无规线团的理论值 0.60。持久长度也相似，除了在 95% DMSO 中较小，这表明在这种溶剂中多肽在局部尺度上更无序。我们展开组中的三种蛋白质具有带电荷残基的不对称序列分布。虽然这些蛋白质在水和 6 M 脲中表现正常，但它们在 40% HFIP 和 95% DMSO 中给出异常低的 R 值，表明它们形成静电发夹，这是由其不对称序列电荷分布和 DMSO 和 HFIP 的低介电常数所促进的。虽然扩散有序 NMR 光谱可以分离小分子，但我们发现许多因素结合在一起，使得蛋白质大小的分子在混合物中更难以分辨。最后，我们研究了表观扩散系数的温度依赖性。小分子表现出线性温度响应，而大蛋白质在高温下由于对流表现出异常大的表观扩散系数，表明扩散参考标准仅在接近 25°C 时才有用。
