Abstract
Membrane proteins are involved in various critical biological processes, and studying membrane proteins represents a major challenge in protein biochemistry. As shown by both structural and functional studies, the membrane environment plays an essential role for membrane proteins. In vitro studies are reliant on the successful reconstitution of membrane proteins. This review describes the interaction between detergents and lipids that aids the understanding of the reconstitution processes. Then the techniques of detergent removal and a few useful techniques to refine the formed proteoliposomes are reviewed. Finally the applications of reconstitution techniques to study membrane proteins involved in Ca(2+) signaling are summarized.
Highlights
Membrane proteins are essential for cellular life
This approach has been employed successfully to study the function of various ion channels and receptors, such as sodium channels [6], potassium channels [7,8], calcium channels [9], and IP3Rs [10] assessed by measuring the movement of radioactive ions/calcium ions, or the change of membrane potentials
No.1 concerning specific reconstitution methods or specific classes of membrane proteins [1,14 22]. It is the purpose of this article to review, after a brief introduction describing the interaction between lipids and detergents and the methods of preparing proteoliposomes, the applications of reconstitution techniques to study membrane proteins involved in Ca2+ signaling
Summary
Reconstitution of membrane proteins into liposomes usually starts with the isolation of cellular membranes. When considering the added effects of other components (salt, pH, etc.), small changes in experimental conditions may give rise to detergent effects not expected from the pure detergent This is why the detergent-mediated reconstitution process is still not fully understood. The reconstitution efficiency depends on the membrane protein of interest, the composition of the lipids, the removal rate of detergent, the ionic conditions and the precise conditions of the initial detergent solubilization. If detergent is removed rapidly, detergent is removed from both binary and ternary micelles, which results in a homogeneous population of vesicles, all having the average lipid-to-protein ratio. If the detergent is removed slowly, it is removed from ternary micelles preferentially, which results in protein-rich vesicles This is supported by the observation that only the population of empty liposomes increased when lipid amount was increased for Ca2+-ATPase reconstitution [28]. The inside-out orientation keeps the large intracellular domain outside of the liposome
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