Sandin Suzanne I, de Alba Eva
Department of Bioengineering, School of Engineering, University of California Merced, Merced, CA 95343, USA.
Chemistry and Biochemistry Ph.D. Program, University of California Merced, Merced, CA 95343, USA.
Methods Protoc. 2022 Feb 16;5(1):19. doi: 10.3390/mps5010019.
Members of the saposin-fold protein family and related proteins sharing a similar fold (saposin-like proteins; SAPLIP) are peripheral-membrane binding proteins that perform essential cellular functions. Saposins and SAPLIPs are abundant in both plant and animal kingdoms, and peripherally bind to lipid membranes to play important roles in lipid transfer and hydrolysis, defense mechanisms, surfactant stabilization, and cell proliferation. However, quantitative studies on the interaction between proteins and membranes are challenging due to the different nature of the two components in relation to size, structure, chemical composition, and polarity. Using liposomes and the saposin-fold member saposin C (sapC) as model systems, we describe here a method to apply solution NMR and dynamic light scattering to study the interaction between SAPLIPs and synthetic membranes at the quantitative level. Specifically, we prove with NMR that sapC binds reversibly to the synthetic membrane in a pH-controlled manner and show the dynamic nature of its fusogenic properties with dynamic light scattering. The method can be used to infer the optimal pH for membrane binding and to determine an apparent dissociation constant (K) for protein-liposome interaction. We propose that these experiments can be applied to other proteins sharing the saposin fold.
鞘脂激活蛋白折叠蛋白家族成员以及具有相似折叠结构的相关蛋白（类鞘脂激活蛋白；SAPLIP）是外周膜结合蛋白，执行重要的细胞功能。鞘脂激活蛋白和SAPLIP在植物和动物界均很丰富，它们在外周与脂质膜结合，在脂质转运与水解、防御机制、表面活性剂稳定以及细胞增殖中发挥重要作用。然而，由于蛋白质和膜这两种成分在大小、结构、化学组成和极性方面性质不同，对它们之间相互作用进行定量研究具有挑战性。我们以脂质体和鞘脂激活蛋白折叠成员鞘脂激活蛋白C（sapC）作为模型系统，在此描述一种应用溶液核磁共振（NMR）和动态光散射在定量水平研究SAPLIP与合成膜之间相互作用的方法。具体而言，我们通过核磁共振证明，sapC在pH可控的情况下与合成膜可逆结合，并通过动态光散射展示其融合特性的动态性质。该方法可用于推断膜结合的最佳pH，并确定蛋白质 - 脂质体相互作用的表观解离常数（K）。我们认为这些实验可应用于其他具有鞘脂激活蛋白折叠结构的蛋白质。
