Structural studies of membrane proteins are still hampered by difficulties of finding appropriate membrane mimicking media that maintain protein structure and function. Phospholipid nanodiscs seem promising to overcome the intrinsic problems of detergent containing environments. While nanodiscs can offer a near native environment, the large particle size complicates their routine use in the structural analysis of membrane proteins by solution NMR. Here, we introduce nanodiscs assembled from shorter ApoA-I protein variants that are of markedly smaller diameter and show that the resulting discs provide critical improvements for the structure determination of membrane proteins by NMR. Using the bacterial outer membrane protein OmpX as an example, we demonstrate that the combination of small nanodisc size, high deuteration levels of protein and lipids and the use of advanced non-uniform NMR sampling methods enable the NMR resonance assignment as well as the high-resolution structure determination of polytopic membrane proteins in a detergent-free, near-native lipid bilayer setting. By applying this method to bacteriorhodopsin we show that our smaller nanodiscs can also be beneficial for the structural characteri-zation of the important class of seven-transmembrane helical proteins. Our set of engineered nanodiscs of subsequently smaller diameters can be used to screen for optimal NMR spectral quality for any given membrane protein while still providing a functional environment. In addi-tion to their key improvements for de novo structure determination, due to their smaller size these nanodiscs enable the investigation of inter-actions between membrane proteins and their (soluble) partner proteins, unbiased by the presence of detergents that might disrupt biological relevant interactions.
"Several manuscripts explore the use on phospholipid nanodiscs for studies of integral membrane proteins. Wagner's group has recently introduced small phospholipid nanodiscs to obtain well-resolved liquid state NMR spectra of membrane proteins (Hagn et al. 2013; Raschle et al. 2009). Here they use these nanodiscs to determine a high-resolution structure of the b-barrel protein OmpX (Hagn and Wagner 2014). "
[Show abstract][Hide abstract] ABSTRACT: The functional importance of proteins that interact with biological membranes can hardly be overestimated. About half of the medicinal drug targets are membrane proteins. Nevertheless, the structural biology of these proteins has been very challenging as only a little more than 500 unique membrane protein structures are present in the PDB (out of 100,000 structures) and also accessible in a membrane protein database (http://blanco.biomol.uci.edu/mpstruc/). About 16 % of these have been derived by NMR spectroscopy. While NMR is limited by molecular size it has the unparalleled capability of observing internal mobility, which can be analyzed at atomic resolution once resonance assignments are obtained. The main obstacles for assignment, dynamics studies and structure determination are low overexpression levels and a high content of hydrophobic amino acids, necessary for embedding into a biological membrane. Thus, some membrane mimetic must always be part of the protein preparation, both ...
"Several types of animal and plant cells are surrounded with a two-layer covering, which is called the phospholipid bilayer . As shown in Figure 2, the molecules that make up the phospholipid bilayer, called phospholipids, organize themselves into two corresponding layers, shaping a covering that can only be infiltrated by certain kinds of substances . This gives the cell an apparent barrier and keeps useless materials out . "
[Show abstract][Hide abstract] ABSTRACT: Graphene is an attention-grabbing material in electronics, physics, chemistry, and even biology because of its unique properties such as high surface-area-to-volume ratio. Also, the ability of graphene-based materials to continuously tune charge carriers from holes to electrons makes them promising for biological applications, especially in lipid bilayer-based sensors. Furthermore, changes in charged lipid membrane properties can be electrically detected by a graphene-based electrolyte-gated graphene field effect transistor (GFET). In this paper, a monolayer graphene-based GFET with a focus on the conductance variation caused by membrane electric charges and thickness is studied. Monolayer graphene conductance as an electrical detection platform is suggested for neutral, negative, and positive electric-charged membrane. The electric charge and thickness of the lipid bilayer (Q
LP and L
LP) as a function of carrier density are proposed, and the control parameters are defined. Finally, the proposed analytical model is compared with experimental data which indicates good overall agreement.
Nanoscale Research Letters 07/2014; 9(1):371. DOI:10.1186/1556-276X-9-371 · 2.78 Impact Factor
"Nevertheless, in the absence of a functional test in vitro, high-quality spectroscopic signals do not prove that the protein is in its native conformation (Poget and Girvin 2007; Zhou and Cross 2013; Catoire et al. 2014). For instance, OmpX exhibits various backbone 15 N/ 1 H N chemical shifts depending on the surfactant used (Fernández et al. 2001; Lee et al. 2008; Hagn et al. 2013). These variations are unlikely to be due to changes in the transmembrane electronic environment, given that, whatever the surfactant used, the amino acids pointing toward the membrane face mostly CH n moieties. "
[Show abstract][Hide abstract] ABSTRACT: Solution-state nuclear magnetic resonance studies of membrane proteins are facilitated by the increased stability that trapping with amphipols confers to most of them as compared to detergent solutions. They have yielded information on the state of folding of the proteins, their areas of contact with the polymer, their dynamics, water accessibility, and the structure of protein-bound ligands. They benefit from the diversification of amphipol chemical structures and the availability of deuterated amphipols. The advantages and constraints of working with amphipols are discussed and compared to those associated with other non-conventional environments, such as bicelles and nanodiscs.
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