Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
Biophys J. 2012 Nov 21;103(10):2203-14. doi: 10.1016/j.bpj.2012.10.012. Epub 2012 Nov 20.
Many intrinsically disordered proteins (IDPs) are significantly unstructured under physiological conditions. A number of these IDPs have been shown to undergo coupled folding and binding reactions whereby they can gain structure upon association with an appropriate partner protein. In general, these systems display weaker binding affinities than do systems with association between completely structured domains, with micromolar K(d) values appearing typical. One such system is the association between α- and β-spectrin, where two partially structured, incomplete domains associate to form a fully structured, three-helix bundle, the spectrin tetramerization domain. Here, we use this model system to demonstrate a method for fitting association and dissociation kinetic traces where, using typical biophysical concentrations, the association reactions are expected to be highly reversible. We elucidate the unusually slow, two-state kinetics of spectrin assembly in solution. The advantages of studying kinetics in this regime include the potential for gaining equilibrium constants as well as rate constants, and for performing experiments with low protein concentrations. We suggest that this approach would be particularly appropriate for high-throughput mutational analysis of two-state reversible binding processes.
许多固有无序蛋白质（IDPs）在生理条件下显著无结构。已经证明其中一些 IDPs 经历偶联折叠和结合反应，通过与适当的伴侣蛋白结合，它们可以获得结构。通常，这些系统的结合亲和力比具有完全结构域之间的关联的系统弱，具有微摩尔 K(d) 值的系统是典型的。这样的系统之一是α-和β- spectrin 之间的关联，其中两个部分结构的、不完整的结构域缔合形成完全结构的、三螺旋束 spectrin 四聚化结构域。在这里，我们使用这个模型系统来演示一种拟合关联和解离动力学轨迹的方法，其中在典型的生物物理浓度下，关联反应预计是高度可逆的。我们阐明了 spectrin 在溶液中组装的异常缓慢的两态动力学。在该区域研究动力学的优点包括获得平衡常数和速率常数的潜力，以及在低蛋白浓度下进行实验的潜力。我们认为，这种方法特别适合于高通量突变分析二态可逆结合过程。
