Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA.
Silicon Therapeutics, Boston, MA, USA.
Methods Mol Biol. 2020;2141:463-475. doi: 10.1007/978-1-0716-0524-0_23.
Nuclear magnetic resonance (NMR) titration and isothermal titration calorimetry can be combined to provide an assessment of how multivalent intrinsically disordered protein (IDP) interactions can involve enthalpy-entropy balance. Here, we describe the underlying technical details and additional methods, such as dynamic light scattering analysis, needed to assess these reactions. We apply this to a central interaction involving the disordered regions of phe-gly nucleoporins (FG-Nups) that contain multiple phenylalanine-glycine repeats which are of particular interest, as their interactions with nuclear transport factors (NTRs) underlie the paradoxically rapid yet also highly selective transport of macromolecules mediated by the nuclear pore complex (NPC). These analyses revealed that a combination of low per-FG motif affinity and the enthalpy-entropy balance prevents high-avidity interaction between FG-Nups and NTRs while the large number of FG motifs promotes frequent FG-NTR contacts, resulting in enhanced selectivity.
核磁共振（NMR）滴定和等温滴定量热法可结合使用，以评估多价固有无序蛋白（IDP）相互作用如何涉及焓熵平衡。在这里，我们描述了评估这些反应所需的基本技术细节和其他方法，例如动态光散射分析。我们将其应用于涉及无序区的中央相互作用phe-gly 核孔蛋白（FG-Nups），其包含多个苯丙氨酸-甘氨酸重复序列，这些重复序列特别有趣，因为它们与核转运因子（NTR）的相互作用是核孔复合物（NPC）介导的大分子快速但又高度选择性运输的基础。这些分析表明，低每个 FG 基序亲和力和焓熵平衡的组合防止了 FG-Nups 和 NTR 之间的高亲和力相互作用，而大量的 FG 基序促进了 FG-NTR 的频繁接触，从而提高了选择性。
