Nonmicellar systems for solution NMR spectroscopy of membrane proteins

Abstract

Integral membrane proteins play essential roles in many biological processes, such as energy transduction, transport of molecules, and signaling. The correct function of membrane proteins is likely to depend strongly on the chemical and physical properties of the membrane. However, membrane proteins are not accessible to many biophysical methods in their native cellular membrane. A major limitation for their functional and structural characterization is thus the requirement for an artificial environment that mimics the native membrane to preserve the integrity and stability of the membrane protein. Most commonly employed are detergent micelles, which can however be detrimental to membrane protein activity and stability. Here, we review recent developments for alternative, nonmicellar solubilization techniques, with a particular focus on their application to solution NMR studies. We discuss the use of amphipols and lipid bilayer systems, such as bicelles and nanolipoprotein particles (NLPs). The latter show great promise for structural studies in near native membranes.

Figure 1

Schematic representation of different solubilization methods for integral membrane proteins. Micelle (a), bicelle (b), reverse micelle (c), amphipol (d), and nanolipoprotein particles (NLP; e). Shown are cartoons depicting each solubilization system. The membrane protein is depicted in gray, with its hydrophilic and hydrophobic regions in light and dark gray, respectively. Detergents are colored in magenta and lipids in green. The amphipol polymer chain is shown as black lines; the negative charges are indicated by minus signs. The apolipoprotein is represented as a blue ring. With the exception of the reverse micelle assembly (c) all systems are in aqueous solution. The reverse micelles (c) are dissolved in organic solvents; areas containing aqueous solution are indicated.

Figure 2

Characterization of human VDAC-1 and bacteriorhodopsin in different membrane mimicking media. 2D [¹⁵N;¹H]-TROSY spectrum of VDAC-1 incorporated in LDAO micelles (a) and in DMPC-nanolipoprotein particles (NLPs, b). Electron micrograph of negatively stained VDAC-1 in DMPC-NLPs (c). 2D [¹⁵N;¹H]-TROSY spectra of bacteriorhodopsin in DDM micelles (d), DMPC-NLPs (e) and A8–35 amphipols (f). Note that the experimental NMR time and protein concentration vary for these samples. In particular, the protein concentrations in the micelle samples were substantially higher than those in the respective non-micellar preparations, resulting in increased quality of TROSY cross peaks in (a) and (d). (panels (a)–(c) reproduced with permission from [47], (d)–(f) unpublished data).