沉降速率法研究蛋白质自聚集

Analysis of protein self-association by sedimentation velocity

分析超速离心(analytical ultracentrifugation,AUC)技术可以记录溶液中不同聚合状态的蛋白质在离心场中的沉降运动轨迹,分析获得其沉降系数分布,进而测定自聚集平衡常数。分析超速离心-沉降速率法(AUC-SV)是分析蛋白质自聚集的经典方法,可以测量从pmol/L到mmol/L的自聚集平衡常数。该实验室与美国国立卫生研究院Peter Schuck团队合作,以常见商用蛋白β-lactoglobulin(βLGB)的同源二聚作为模型,准确测定了其自聚集平衡常数Kd。文章详细介绍了蛋白质自聚集实验中沉降速率实验设计、数据分析的操作步骤,建立了一套应用沉降速率法研究蛋白质自聚集的标准方法,并利用该方法对脱落酸(ABA)受体蛋白PYL3,以及结合ABA的PYL3的自聚集平衡常数进行测定,进一步验证了该方法的可靠性。

Analytical ultracentrifugation(AUC)technology can record the sedimentation trajectory of proteins in different polymerization states in solution in the centrifugal field,by obtaining the distribution of sedimentation coefficient to study the self-association equilibrium constant.Analytical ultracentrifugation-sedimentation velocity(AUC-SV)is a classical method for analyzing self-association equilibrium constant from pmol/L to mmol/L.In collaboration with Peter Schuck’s group at the USA National Institutes of Health,the self-association equilibrium constant Kd of commercially available protein β-lactoglobulin(βLGB)as a proof of principle is accurately determined.This paper introduces in detail the operation steps of sedimentation velocity experimental design and data analysis in protein self-association experiment,and establishes a set of standard methods for studying protein self-association by sedimentation velocity method.The self-association equilibrium constants of abscisic acid(ABA)receptor protein PYL3 and ABA bound PYL3 are determined by this method,which further verified the reliability of it.