Abstract
We report the optimization of detergent-mediated reconstitution of an integral membrane-bound protein, full-length influenza M2 protein, by direct insertion into detergent-saturated liposomes. Detergent-mediated reconstitution is an important method for preparing proteoliposomes for studying membrane proteins, and must be optimized for each combination of protein and membrane constituents used. The purpose of the reconstitution was to prepare samples for site-directed spin-labeling electron paramagnetic resonance (SDSL-EPR) studies. Our goals in optimizing the protocol were to minimize the amount of detergent used, reduce overall proteoliposome preparation time, and confirm the removal of all detergent. The liposomes were comprised of (1-palmitoyl-2-oleyl-sn-glycero-phosphocholine (POPC) and 1-palmitoyl-2-oleyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG), and the detergent octylglucoside (OG) was used for reconstitution. Rigorous physical characterization was applied to optimize each step of the reconstitution process. We used dynamic light scattering (DLS) to determine the amount of OG needed to saturate the preformed liposomes. During detergent removal by absorption with Bio-Beads, we quantified the detergent concentration by means of a colorimetric assay, thereby determining the number of Bio-Bead additions needed to remove all detergent from the final proteoliposomes. We found that the overnight Bio-Bead incubation used in previously published protocols can be omitted, reducing the time needed for reconstitution. We also monitored the size distribution of the proteoliposomes with DLS, confirming that the size distribution remains essentially constant throughout the reconstitution process.
Highlights
Membrane proteins play a critical and often multifaceted role in many biophysical processes.Approximately one third of human genes code for membrane proteins [1]; because membrane proteins are implicated in many diseases, they are the subject of much current research [2]
We report the optimization and detailed, rigorous physical characterization of detergent-mediated reconstitution by direct insertion into pre-formed liposomes of an integral membrane protein that is of considerable current interest, namely influenza M2 protein [12,13]
We found that the average effective diameter of the liposome distribution remains essentially constant throughout the process, confirming that direct insertion of the protein in detergent-saturated liposomes maintains the liposome size distribution
Summary
Membrane proteins play a critical and often multifaceted role in many biophysical processes.Approximately one third of human genes code for membrane proteins [1]; because membrane proteins are implicated in many diseases, they are the subject of much current research [2]. Membrane proteins play a critical and often multifaceted role in many biophysical processes. Many studies of membrane proteins involve reconstituting the proteins into lipid bilayer vesicles, in order to preserve the structure and function of these proteins for studies of structure and/or function in a native-like. Reproducible methods for the reconstitution of purified membrane proteins into model membranes have been a long-standing challenge [4,5,6,7,8,9,10,11]. The success of a reconstitution protocol is sensitive to the protein, the type of lipids used, and the choice of detergent [6]. The optimization of the reconstitution strategy for each new experimental system is necessary for the collection of reproducible high-quality data
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