Solid-State NMR Study to Probe the Effects of Divalent Metal Ions (Ca²⁺ and Mg²⁺) on the Magnetic Alignment of Polymer-Based Lipid Nanodiscs

Abstract

Divalent cations, especially Ca2+ and Mg2+, play a vital role in the function of biomolecules and making them important to be constituents in samples for in vitro biophysical and biochemical characterizations. Although lipid nanodiscs are becoming valuable tools for structural biology studies on membrane proteins and for drug delivery, most types of nanodiscs used in these studies are unstable in the presence of divalent metal ions. To avoid the interaction of divalent metal ions with the belt of the nanodiscs, synthetic polymers have been designed and demonstrated to form stable lipid nanodiscs under such unstable conditions. Such polymer-based nanodiscs have been shown to provide an ideal platform for structural studies using both solid-state and solution NMR spectroscopies because of the near-native cell-membrane environment they provide and the unique magnetic-alignment behavior of large-size nanodiscs. In this study, we report an investigation probing the effects of Ca2+ and Mg2+ ions on the formation of polymer-based lipid nanodiscs and the magnetic-alignment properties using a synthetic polymer, styrene maleimide quaternary ammonium (SMA-QA), and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids. Phosphorus-31 NMR experiments were used to evaluate the stability of the magnetic-alignment behavior of the nanodiscs for varying concentrations of Ca2+ or Mg2+ at different temperatures. It is remarkable that the interaction of divalent cations with lipid headgroups promotes the stacking up of nanodiscs that results in the enhanced magnetic alignment of nanodiscs. Interestingly, the reported results show that both the temperature and the concentration of divalent metal ions can be optimized to achieve the optimal alignment of nanodiscs in the presence of an applied magnetic field. We expect the reported results to be useful in the design of nanodisc-based nanoparticles for various applications in addition to atomic-resolution structural and dynamics studies using NMR and other biophysical techniques.

Characterization of Divalent Cation Interactions with AASTY Nanodiscs

“…This comes at a cost of well-formed nanodiscs. An explanation could be the rouleaux stacking of discs observed by Ravula et al under similar conditions . Notably, AASTY 7.4 -52 is least affected by the presence of divalent cations both in nanodiscs and in its free form, and while all nanodiscs tolerate the presence of 3 mM CaCl 2 reasonably well, their behaviors start to diverge significantly beyond this concentration (Figure D).…”

“…Recently, Ravula et al used NMR to characterize the effects of Ca 2+ and Mg 2+ ions finding rouleaux stacking of nanodiscs with divalent cations. 27 This finding further highlights the benefit of in-depth characterization of these systems. An often cited disadvantage of styrene based copolymers such as AASTY and SMA is their strong absorption of UV light overlapping with the absorption of proteins at 280 nm, which is commonly used for the determination of protein concentration and purity.…”

“…Lipid-associated copolymer in nanodiscs has reduced degrees of freedom compared to the free copolymer, which could cause the nanodiscs to stick together and aggregate at lower Ca 2+ concentrations, while divalent cation binding to the lipid headgroups could cause rouleaux stacking of nanodiscs as observed by Ravula et al. by NMR …”

“…Furthermore, as these techniques record properties of an ensemble of species in solution, the signal from nanodiscs and free copolymer remaining in the solution cannot be separated from one another. Recently, Ravula et al used NMR to characterize the effects of Ca 2+ and Mg 2+ ions finding rouleaux stacking of nanodiscs with divalent cations . This finding further highlights the benefit of in-depth characterization of these systems.…”

Measurement of Residual Dipolar Couplings Using Magnetically Aligned and Flipped Nanodiscs

“…The need for atomic-resolution structures of membrane proteins has been well demonstrated by recent studies. − While there are many challenges in obtaining near-native structures, the introduction of nanodiscs has opened new avenues for structural and functional studies of membrane proteins. − A nanodisc is a lipid bilayer disc surrounded by an amphipathic molecule such as membrane scaffold protein (MSP), DNA, short amphipathic peptides, − or synthetic amphipathic polymers. − While all of these nanodiscs have been demonstrated to be excellent membrane mimetics and quite valuable for membrane protein structural biology, polymer-based nanodisc studies have expanded the flexibility and scope of the nanodisc technology as synthetic polymers can be tailored to the need. ,− The recent developments of different types of synthetic polymers have enhanced the applications of polymer-based nanodiscs by making them amenable for investigation by a variety of biophysical techniques including solution and solid-state NMR spectroscopy and cryoEM. ,− One of the unique advantages of these polymer nanodiscs is that polymers can be used to directly extract membrane proteins along with lipids from their cellular environment without the use of a detergent. , As a result, it is possible to functionally reconstitute a membrane protein in a native lipid environment for further structural and functional studies. Another advantage is that the size of polymer nanodiscs can be varied by simply changing the ratio between the polymer and lipids. , The small-size nanodiscs (<20 nm diameter) are useful for well-established solution NMR studies, while large-size nanodiscs (>20 nm in diameter, also called as “macro–nanodiscs”) magnetically align and therefore useful for static solid-state NMR studies. , These magnetically aligned macro–nanodiscs have also been demonstrated to be useful in the measurement of anisotropic NMR parameters such as residual dipolar couplings (RDCs) and residual quadrupolar couplings (RQCs) .…”