ABSTRACT
Affinity tags are valuable tools for high-throughput protein isolation in automated screenings or downstream processing approaches and are also widely used in laboratory applications for quick and easy access to many proteins. Here, we describe the preparative purification of soluble extended synaptotagmin 2 (rE-Syt2) at bench scale for basic structural and functional studies. Due to the low protein stability, a classical purification procedure without affinity tag was more powerful than isolation of His((6))-tagged rE-Syt2 and subsequent proteolytic tag-removal. Furthermore, expression analysis of truncated rE-Syt2 variants suggested a concept of interdependent-domain organization in proteins containing multiple C2 domains.
Subject(s)
Chromatography/methods , Gene Expression , Synaptotagmins/chemistry , Synaptotagmins/isolation & purification , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Histidine/chemistry , Histidine/genetics , Histidine/isolation & purification , Histidine/metabolism , Oligopeptides/chemistry , Oligopeptides/genetics , Oligopeptides/isolation & purification , Oligopeptides/metabolism , Protein Stability , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Solubility , Synaptotagmins/genetics , Synaptotagmins/metabolism , Thrombin/metabolismABSTRACT
The protein family of membrane-anchored extended synaptotagmin-like proteins (E-Syts) was recently discovered in humans. E-Syt1 to 3 each contain at least one transmembrane domain and three or five C2 domains. To investigate the whole C2 area of murine E-Syt2, highly pure recombinant E-Syt2 (rE-Syt2) covering all three C2 domains was isolated. The structure of rE-Syt2 was studied by small-angle X-ray scattering (SAXS) providing a three-dimensional image of a protein with three C2 domains. Calcium binding of rE-Syt2 triggered structural rearrangements and initiated reversible multimerization of the protein in vitro. Quantitative analysis of the calcium binding revealed an apparent binding constant of 100muM. This is the first structural study of a multi-C2 protein, presumably involved in Ca-dependent signalling events.
Subject(s)
Calcium-Binding Proteins/chemistry , Membrane Proteins/chemistry , Models, Molecular , Animals , Calcium/chemistry , Calcium-Binding Proteins/classification , Calcium-Binding Proteins/genetics , Conserved Sequence , Humans , Membrane Proteins/classification , Membrane Proteins/genetics , Mice , Phylogeny , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Scattering, Small Angle , Solubility , X-Ray DiffractionABSTRACT
The generation of autoantibodies against chromatin is a hallmark of the multifactorial autoimmune disease systemic lupus erythematosous (SLE). Impaired clearance of apoptotic cells together with the release of nuclear autoantigens are supposed to contribute to the loss of self-tolerance in SLE. Phospholipids such as phosphatidylserine (PS) and phosphatidylethanolamine (PE) are exposed on the surfaces of apoptotic cells and on apoptotic blebs. Also histones/nucleosomes can be detected on apoptotic cells; however, their binding motifs are still unknown. Therefore, we investigated the interaction of PS, PE, phosphatidylcholine (PC), and cardiolipin (CL) with histones H1, H2A, H2B, H3, and H4 by surface plasmon resonance (SPR). Strong binding to phospholipids was found for all histones, with H2A displaying the highest binding affinity to all phospholipids investigated. Hence, phospholipids including PS and PE may contribute to the binding of histones to surfaces and blebs of apoptotic cells. Moreover, histones/nucleosomes complexed to uningested apoptotic membrane structures may foster autoimmunity towards nuclear compounds.
