Tailored nanodisc particles for structural and functional studies of membrane proteins

Structural studies of membrane proteins are still hampered by difficulties in finding appropriate membrane mimicking media that maintain protein structure and function. A novel membrane system, called phospholipid nanodiscs, consisting of a patch of lipids surrounded by two copies of a lipid-binding protein, recently became popular in the membrane protein field. In this system, membrane proteins can be investigated in a real detergent-free and native-like lipid environment. Smaller nanodiscs tailored to accommodate membrane proteins of different sizes are now available to facilitate the structure determination process of membrane proteins with NMR spectroscopy. This development provides a route to obtaining three-dimensional structural information about membrane proteins in a realistic environment and enables the experimental determination of protein dynamics in a lipid bilayer setting that is often correlated to their functional properties.

HFSP Long-Term Fellow Franz Hagn and colleagues
authored on Thu, 04 April 2013

Compared to the plethora of structural data on soluble proteins, integral membrane proteins are far less well studied, both functionally and structurally. This is primarily due to difficulties in producing integral membrane proteins in folded and active forms as well as in sufficient yields.  A major part of this problem is the selection of an appropriate membrane-mimicking environment supporting both function and stability of a particular membrane protein. Usually detergents are employed for membrane protein preparation and detergent micelles are the most common media for structural investigations. However, a pure detergent environment may lower membrane protein stability, and abolish their biological function. Even detergent solubilized membrane proteins often contain bound lipids emphasizing the beneficial effect of a lipid environment for their structure and stability. Furthermore, detergents can hamper interaction studies between membrane proteins and soluble factors, as they tend to destabilize or even denature soluble interaction partners. The use of phospholipid bilayers can overcome these problems, as they resemble a considerably more native membrane environment. In addition, phospholipid bilayers can be prepared to match any desired lipid composition required to mimic the native environment and thus stabilize the respective membrane protein. Despite the apparent advantages of nanodiscs, no structure of a membrane protein inserted into this novel membrane mimic was available so far. This might be due to problems in crystallizing these particles for X-ray analysis, but also due to the large size of the available nanodiscs that excludes them from an analysis by NMR.

HFSP Long-Term Fellow, Franz Hagn, working with Gerhard Wagner at Harvard Medical School, has now succeeded in designing novel variants of apolipoproteinA-1 (ApoA-1) that assemble into nanodiscs of subsequently smaller diameters. With this set of nanodiscs it is now possible to adjust the diameter of a nanodisc to the size for the protein of interest without adding too much in molecular weight to the system, which might complicate NMR analysis. Due to size limitations, this is a critical feature in conducting NMR structure determination of membrane proteins. Using smaller nanodiscs and advanced NMR methods, the authors could determine the structure of the bacterial outer membrane protein OmpX at high resolution. Furthermore, they were able to determine the dynamical properties of this protein in nanodiscs by NMR relaxation experiments. This experimental dynamical data compares well with molecular dynamics simulations of OmpX conducted in a lipid bilayer environment.

Figure: Size-optimized phospholipid nanodiscs for structural studies of membrane proteins with NMR spectroscopy. Nanodiscs consist of lipids encircled by two copies of apolipoprotein A-1 (ApoA-1). The length of the ApoA-1 defines the diameter of the nanodisc. Truncated ApoA-1 constructs lead to smaller nanodiscs as monitored by negative-stain electron microscopy (EM), which are suitable for high-resolution structure determination by NMR spectroscopy.

This is the first example of a membrane protein structure solved in phospholipid nanodiscs. The use of the introduced smaller nanodiscs was a critical step in this process and enabled a detailed NMR spectroscopic characterization of OmpX in a phospholipid bilayer system. The use of a native-like membrane environment might be even more important for marginally stable membrane proteins, which so far eluded structural studies due to limited stability in detergent micelles. Phospholipid nanodiscs may turn out to be an ideal choice for maintaining longer-term stability for structural studies, preserving a particular membrane protein in its functional form and enabling binding studies in a detergent free environment. With the smaller MSP variants reported by the authors, solution NMR spectroscopy shows great promise in addressing important functional and structural questions about membrane proteins in their native lipid bilayer environment.   

Reference

Optimized phospholipid bilayer nanodiscs facilitate high-resolution structure determination of membrane proteins. Franz Hagn, Manuel Etzkorn, Thomas Raschle, and Gerhard Wagner, J.Am.Chem.Soc. (2013) 135(5), 1919-1925. [doi: 10.1021/ja310901f]

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