Lebowitz Jacob, Lewis Marc S, Schuck Peter
Molecular Interactions Resource, Division of Bioengineering and Physical Science, ORS, OD, National Institutes of Health, Bethesda, Maryland 20892, USA.
Protein Sci. 2002 Sep;11(9):2067-79. doi: 10.1110/ps.0207702.
Analytical ultracentrifugation (AU) is reemerging as a versatile tool for the study of proteins. Monitoring the sedimentation of macromolecules in the centrifugal field allows their hydrodynamic and thermodynamic characterization in solution, without interaction with any matrix or surface. The combination of new instrumentation and powerful computational software for data analysis has led to major advances in the characterization of proteins and protein complexes. The pace of new advancements makes it difficult for protein scientists to gain sufficient expertise to apply modern AU to their research problems. To address this problem, this review builds from the basic concepts to advanced approaches for the characterization of protein systems, and key computational and internet resources are provided. We will first explore the characterization of proteins by sedimentation velocity (SV). Determination of sedimentation coefficients allows for the modeling of the hydrodynamic shape of proteins and protein complexes. The computational treatment of SV data to resolve sedimenting components has been achieved. Hence, SV can be very useful in the identification of the oligomeric state and the stoichiometry of heterogeneous interactions. The second major part of the review covers sedimentation equilibrium (SE) of proteins, including membrane proteins and glycoproteins. This is the method of choice for molar mass determinations and the study of self-association and heterogeneous interactions, such as protein-protein, protein-nucleic acid, and protein-small molecule binding.
分析超速离心法（AU）正重新成为研究蛋白质的一种多功能工具。监测大分子在离心场中的沉降，可在不与任何基质或表面相互作用的情况下，对其在溶液中的流体动力学和热力学特性进行表征。新型仪器与强大的数据分析计算软件相结合，推动了蛋白质及蛋白质复合物表征方面的重大进展。新进展的速度之快，使得蛋白质科学家难以获得足够的专业知识来将现代分析超速离心法应用于他们的研究问题。为解决这一问题，本综述从蛋白质系统表征的基本概念入手，介绍了先进方法，并提供了关键的计算和网络资源。我们将首先探讨通过沉降速度（SV）对蛋白质进行的表征。沉降系数的测定有助于对蛋白质和蛋白质复合物的流体动力学形状进行建模。已实现对沉降速度数据进行计算处理以解析沉降成分。因此，沉降速度在鉴定寡聚状态和异质相互作用的化学计量方面非常有用。本综述的第二大部分涵盖蛋白质的沉降平衡（SE），包括膜蛋白和糖蛋白。这是测定摩尔质量以及研究自缔合和异质相互作用（如蛋白质 - 蛋白质、蛋白质 - 核酸和蛋白质 - 小分子结合）的首选方法。
