Abstract
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 (KDapp) for protein-liposome interaction. We propose that these experiments can be applied to other proteins sharing the saposin fold.
Highlights
Saposins, known as SAPs, are lysosomal proteins that activate enzymes involved in lipid degradation [1]
The saposin fold is shared by other proteins known as saposin-like proteins (SAPLIPs) displaying a variety of functions that require peripheral membrane binding [4]
The liposome size determined by Dynamic Light Scattering (DLS) can be considered as the diameter of the outer membrane size and the inner membrane can be calculated as indicated in Equation (1), with the assumption that the lipid bilayer is 4 nm
Summary
Known as SAPs (sphingolipid activator proteins), are lysosomal proteins that activate enzymes involved in lipid degradation [1]. The saposin fold is shared by other proteins known as saposin-like proteins (SAPLIPs) displaying a variety of functions that require peripheral membrane binding [4]. Our lab has recently engineered by recombinant technologies a protein conjugate of sapC with the active domain (BH3) of the pro-apoptotic protein PUMA from the Bcl-2 family [6] The purpose of this protein chimera (sapC-PUMA) is to retain sapC functionality and simultaneously improve cytotoxicity in cancer cells via apoptosis [6]. High-resolution NMR techniques were originally used to determine the three-dimensional structure of human saposin C in solution and bound to micelles [2,3], representing the protein conformational change from the closed-soluble form to the open micelle-bound form (Figure 1). When considering differences in size such as those described here for SAPLIPs and liposomes, sity
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