Bitan Gal
UCLA, Department of Neurology, David Geffen School of Medicine, Los Angeles, CA, USA.
Methods Enzymol. 2006;413:217-36. doi: 10.1016/S0076-6879(06)13012-8.
Oligomers of amyloidogenic proteins are believed to be key effectors of cytotoxicity and cause a variety of amyloid-related diseases. Dissociation or inhibition of formation of the toxic oligomers is thus an attractive strategy for the prevention and treatment of these diseases. In order to develop reagents capable of inhibiting protein oligomerization, the structures and mechanisms of oligomer formation must be understood. However, structural studies of oligomers are difficult because of the metastable nature of the oligomers and their existence in mixtures with monomers and other assemblies. A useful method for characterization of oligomer size distributions in vitro is photo-induced cross-linking of unmodified proteins (PICUP) (Fancy and Kodadek, 1999). By providing "snapshots" of dynamic oligomer mixtures, PICUP enables quantitative analysis of the relations between primary and quaternary structures, offering insights into the molecular organization of the oligomers. This chapter discusses the photochemical mechanism; reviews the scope, usefulness, and limitations of PICUP for characterizing metastable protein assemblies; and provides detailed experimental instructions for performing PICUP experiments.
淀粉样蛋白的寡聚体被认为是细胞毒性的关键效应物，并引发多种淀粉样蛋白相关疾病。因此，解离或抑制毒性寡聚体的形成是预防和治疗这些疾病的一种有吸引力的策略。为了开发能够抑制蛋白质寡聚化的试剂，必须了解寡聚体形成的结构和机制。然而，由于寡聚体的亚稳态性质以及它们与单体和其他聚集体混合存在，对寡聚体的结构研究很困难。一种用于体外表征寡聚体大小分布的有用方法是光诱导未修饰蛋白质交联（PICUP）（Fancy和Kodadek，1999年）。通过提供动态寡聚体混合物的“快照”，PICUP能够对一级结构和四级结构之间的关系进行定量分析，从而深入了解寡聚体的分子组织。本章讨论光化学机制；回顾PICUP在表征亚稳态蛋白质聚集体方面的范围、有用性和局限性；并提供进行PICUP实验的详细实验说明。
