Structures and Interactions of Transmembrane Targets in Native Nanodiscs

Abstract

Transmembrane proteins function within a continuous layer of biologically relevant lipid molecules that stabilizes their structures and modulates their activities. Structures and interactions of biological membrane–protein complexes or “memteins” can now be elucidated using native nanodiscs made by poly(styrene co-maleic anhydride) derivatives. These linear polymers contain a series of hydrophobic and polar subunits that gently fragment membranes into water-soluble discs with diameters of 5–50 nm known as styrene maleic acid lipid particles (SMALPs). High-resolution structures of memteins that include endogenous lipid ligands and posttranslational modifications can be resolved without resorting to synthetic detergents or artificial lipids. The resulting ex situ structures better recapitulate the in vivo situation and can be visualized by methods including cryo-electron microscopy (cryoEM), electron paramagnetic resonance (EPR), mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, small angle x-ray scattering (SAXS), and x-ray diffraction (XRD). Recent progress including 3D structures of biological bilayers illustrates how polymers and native nanodiscs expose previously inaccessible membrane assemblies at atomic resolution and suggest ways in which the SMALP system could be exploited for drug discovery.

Magnetic-Alignment of Polymer Nanodiscs Probed by Solid-State NMR Spectroscopy

“…Further increase in the concentration of YbCl3 did not show any difference in the 31 P peak intensity indicating no major changes in the alignment of macro-nanodiscs. 31 P NMR spectra of unpurified SMA-QA:DMPC nanodiscs recorded as a function of time at 308 K, and at various concentrations of YbCl3. 31 P peaks appearing at ~ -15, 0 and -22 ppm indicate a perpendicular orientation, isotropic phase, and a parallel orientation, respectively.…”

“…In fact, for a range of temperatures (between 295 and 310 K), the 31 P NMR spectra exhibited no isotropic behavior indicating an alignment of all macro-nanodiscs in the sample. 31 P NMR spectra were also recorded upon the addition of different concentrations of YbCl3 as a function of temperature (Figure 3). For 0.5 and 1 mM concentrations of YbCl3, 31 P NMR spectra showed a combination of powder pattern, isotropic peak, alignment with the bilayer normal perpendicular to the magnetic field direction, and flipping of nanodiscs with the bilayer normal parallel to the magnetic field direction depending on the temperature of the sample as shown in Figure 3.…”

“…These behavior of macro-nanodiscs is similar to that of that of the well-studied bicelles samples. [47][48] 31 P NMR spectra of DMPC:SMA-QA (1:0.5 w/w) macro-nanodiscs in the absence (column 1) and presence of YbCl3 (columns 2-4) at different temperatures. A 100 mg/mL lipid concentration was used in the sample.…”

“…[25][26] Recent developments in the polymer nanodiscs field expanded the applications of nanodiscs by using a wide variety of biophysical techniques to study membrane proteins. 17,19,[27][28][29][30][31][32][33][34][35][36][37][38][39] For accommodation of different sizes of membrane proteins and membrane associated proteinprotein complexes, and to achieve magnetic-alignment, the size of nanodiscs should be variable. This is shown to be achievable with different polymer to lipid ratios.…”

“…P NMR experiments: Time dependent NMR experiments were performed on an Agilent NMR spectrometer operating at the resonance frequency of 699.88 MHz for 1 H and 283.31 MHz for 31 P nuclei. A 4 mm triple-resonance HXY MAS NMR probe (Agilent) was used under static condition.…”