Circular Nanodisk Research Articles

Comparative Analysis of Two Different MIM Configurations of a Plasmonic Nanoantenna

AbstractTwo plasmonic nanoantenna configurations—nanodisk and nanostrip arrays—in a metal–insulator-metal (MIM) setup were proposed, optimized, and compared by simulating their optical properties in three-dimensional models using COMSOL Multiphysics software. The optical responses, including electric field enhancement, absorption, reflection, and transmission spectra, were systematically investigated. Optimized geometrical parameters led to a significant enhancement of the electric field within the gap layers and almost perfect light absorptance for both structures. The results showed that the enhancement of the electric field depends on the polarization of the incident light. For both polarizations, the periodic circular nanodisk array showed a stronger field enhancement with an electric field enhancement factor of 6.6 × 106 and TE polarization, and a larger absorptance of 98% at its dipole resonance wavelength, indicating the fundamental plasmonic mode. In addition, weaker resonant modes were observed in the absorptance and reflectance spectra of both nanostructures, with the nanostrips exhibiting sharper and stronger higher-order modes, making them suitable for applications requiring precise wavelength selectivity and narrow-band responses. Despite their different geometric shapes, both structures exhibited similar optimized metal film thickness and nanoparticle height, comparable modes in number and position, and identical optimized light incidence angles. Furthermore, increasing the dielectric gap layer thickness and optimizing it to a specific value revealed its ability to measure the refractive index, making it a promising candidate for sensing applications.

Abstract 489: Unlocking immune resistance and metastasis of PDAC via dual acting circularized nanodisc

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and deadliest forms of cancer. While major progress in immunotherapy has been made in recent years, it remains poorly effective in this malignancy. Two key players stand behind immune resistance of PDAC and inadequate efficacy of immunotherapy to suppress its metastasis. First, the immune-resistant pancreatic cancer stem cells (CSCs) evade immune surveillance acting as a source for tumor relapse and metastasis. This immune resistance stems from low immunogenicity of PDAC cells as well as the inaccessible CSCs that reside deep in the tumor core. Second, pancreatic cancer associated fibroblasts (CAFs) fuel metastasis of PDAC cells by suppressing their immune recognition and establishing pre-metastatic niche at distant organs to help their colonization. Our hypothesis is to overcome immune resistance of PDAC and inhibit early steps of its metastasis via simultaneous immunogenic eradication of immune resistant CSCs and switching immunosuppressive and pro-metastatic CAFs into quiescent ones. Therefore, we engineered ultrasmall circularized nanodiscs (cND) that encapsulate oncolytic peptide LTX-315 and fibroblast remodeling Notch signaling inhibitor. The engineered oncolytic LTX-315 loaded cND successfully induced immunogenic cell death (ICD) of both bulk PANC1 and pancreatic CSCs. Compared to non-treated and free LTX-315 treated cells, LTX-315 cND resulted in enhanced release of DAMPs including ATP and HMGB-1 as well as surface translocation of calreticulin. The ultrasmall size (9 nm) of cND enabled its deep tumor penetration to eradicate resistant CSCs, as revealed using 3D PDAC heterospheroids. In parallel, the Notch blocking cND promoted quiescence of the activated pancreatic stellate cells as denoted by downregulation of SMA, FAP and collagen. In KPC syngeneic PDAC mouse model, the dual oncolytic/Notch blocking cND combined with aPD-L1 therapy significantly suppressed tumor growth and prevented its metastasis to liver compared to vehicle-treated mice. Immunophenotyping study showed marked reduction in the abundance of CSCs and CAFs in the tumor treated with the cNDs. Moreover, the tumor infiltration of CD86+ MHCII+ dendritic cells and cytotoxic CD8+ T cells was enhanced after treatment with the cND. Overall, our dual strategy of targeting pancreatic CSCs and CAFs via oncolytic/Notch blocking cND boosted the anti-tumor and anti-metastatic efficacy of aPD-L1 therapy to eliminate primary PDAC tumor and suppress its metastasis. Citation Format: Ahmed Elzoghby, Hagar Emam, Seungbin Yim, Cuiyan Xin, Mahmoud Nasr. Unlocking immune resistance and metastasis of PDAC via dual acting circularized nanodisc [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 489.

The Advanced Properties of Circularized MSP Nanodiscs Facilitate High-resolution NMR Studies of Membrane Proteins

Membrane mimetics are essential for structural and functional studies of membrane proteins. A promising lipid-based system are phospholipid nanodiscs, where two copies of a so-called membrane scaffold protein (MSP) wrap around a patch of lipid bilayer. Consequently, the size of a nanodisc is determined by the length of the MSP. Furthermore, covalent MSP circularization was reported to improve nanodisc stability. However, a more detailed comparative analysis of the biophysical properties of circularized and linear MSP nanodiscs for their use in high-resolution NMR has not been conducted so far. Here, we analyze the membrane fluidity and temperature-dependent size variability of circularized and linear nanodiscs using a large set of analytical methods. We show that MSP circularization does not alter the membrane fluidity in nanodiscs. Further, we show that the phase transition temperature increases for circularized versions, while the cooperativity decreases. We demonstrate that circularized nanodiscs keep a constant size over a large temperature range, in contrast to their linear MSP counterparts. Due to this size stability, circularized nanodiscs are beneficial for high-resolution NMR studies of membrane proteins at elevated temperatures. Despite their slightly larger size as compared to linear nanodiscs, 3D NMR experiments of the voltage-dependent anion channel 1 (VDAC1) in circularized nanodiscs have a markedly improved spectral quality in comparison to VDAC1 incorporated into linear nanodiscs of a similar size. This study provides evidence that circularized MSP nanodiscs are a promising tool to facilitate high-resolution NMR studies of larger and challenging membrane proteins in a native lipid environment.

Thermal stability analysis of functionally graded non-uniform asymmetric circular and annular nano discs: Size-dependent regularity and boundary conditions

In this article, the size-dependent thermal buckling analysis of nonuniform functionally graded asymmetric circular and annular nanodiscs is presented on the basis of Kirchhoff's plate theory, Eringen's nonlocal elasticity theory, and physical neutral plane. For the first time, the nonlocal regularity conditions and boundary conditions are obtained for the asymmetric discs. The thickness of the nanodiscs is assumed to be varying linearly and parabolically in the radial direction. The Power-law model is adopted to compute the temperature-independent effective mechanical properties of the functionally graded materials (FGMs). The size-dependent stability equation is obtained from Euler-Lagrange's equation which is derived from Hamilton's principle. This equation and corresponding boundary conditions are discretized by the differential quadrature method (DQM) and provide an eigenvalue problem. The numerical value of the lowest eigenvalue is reported as a critical temperature difference on the surfaces of the nanodiscs. The effects of various parameters such as nonlocal parameter, volume fraction index, nodal lines, and taper parameters for thickness variations are studied.

Feeling at home: Structure of the NTSR1-Gi complex in a lipid environment.

The interaction of G protein–coupled receptors (GPCRs) with heterotrimeric G proteins plays a critical role in signal transduction processes, and multiple GPCR–G protein complexes reconstituted in detergent micelles have been visualized using cryo-EM. A new study reports the structure of neurotensin receptor 1 (NTSR1) in complex with the heterotrimeric Gi protein, assembled in a lipid environment using circularized nanodiscs. The structure sheds light on how the lipid context may influence receptor–G protein coupling and activation.

ZrN fractal-graphene-based metamaterial absorber in the visible and near-IR regimes

The absorption characteristics of zirconium nitride (ZrN)-based metamaterial absorber of fractal geometry are studied. The proposed absorber is comprised of fractal metasurface at the top having subwavelength-sized periodic pattern of specially designed ZrN circular nano-discs arranged over silicon dioxide (SiO2) substrate. A tri-layer graphene, owing to its exhibiting better tunability, is introduced at the interface of metasurface and substrate. The bottom side of SiO2 is coated with silver nanolayer to block transmission. The absorptivity essentially depends on the kind of fractal design used in metasurface to configure the absorber. The obtained results exhibit the absorption characteristics to remain insensitive to the incidence polarizations. The chemical potential of graphene could be used to tune the spectral characteristics. Such a kind of absorber would be useful for filtering, antireflection coating, and solar energy harvesting applications in the visible and near-infrared regimes of electromagnetic spectrum.

Tunable microwave properties of a skyrmion in an isolated nanodisk

A magnetic skyrmion is a topological object having particle-spin like configuration, which can be stabilized in ferromagnetic systems with Dzyaloshinskii-Moriya interaction. In this report, we investigate tunable microwave properties of magnetic skyrmions in a circular nanodisk using micromagnetic simulations. An in-plane ac magnetic field excites gyrotropic dynamics with a clockwise and a counter-clockwise mode. We have shown the dependence of microwave responses on the size of the skyrmion by tuning the Dzyaloshinskii-Moriya interaction, magnetic anisotropy, and out-of-plane external bias magnetic field. A remarkably large frequency shift of 4.2 GHz has been observed for a change of 40 mT bias field. Furthermore, a broad microwave tunability of 10.8 GHz has been shown with a variation of magnetic anisotropy of 1 × 105 J/m3. The manifestation of inertial mass of the skyrmion, its dependence on the resonance responses and the sizes of the skyrmion are explained by using Thiele’s equation. The inertial mass of the skyrmion is estimated to be in the order of 10−23 kg. The results open up the potential use of the skyrmions in reconfigurable nano-magnonic devices with large tunability.

Tunable THz Switch-Filter Based on Magneto-Plasmonic Graphene Nanodisk

We propose and analyze a new multifunctional THz device that can operate as a tunable switch and a tunable filter. The device consists of a circular graphene nanodisk coupled to two nanoribbons oriented at 90° to each other. The graphene elements are placed on a dielectric substrate. The nanodisk is magnetized by a dc magnetic field normal to its plane. The physical principle of the device is based on the propagation of surface plasmon-polariton (SPP) waves in the graphene nanoribbons and excitation of dipole mode in the nanodisk resonator. Numerical simulations show that 0.61 T dc magnetic field provides transmission (regime ON) at the frequency 5.33 THz with the bandwidth (BW) 12.7% and filtering with a Q-factor of 7.8. At the central frequency, the insertion loss (IL) is around -2 dB, and the reflection coefficient is -43 dB. The regime OFF can be achieved by means of switching the dc magnetic field to zero value or by switching the chemical potential of the nanodisk to zero. This results in the ON/OFF ratio better than 20 dB. A small central frequency tuning of the switch by chemical potential is possible with a fixed dc magnetic field.

Control of magnetic vortex circulation in one-side-flat nanodisk pairs by in-plane magnetic filed

In a nanodisk made of soft ferromagnet, the magnetic vortex structure are highly stabilized, and the circulation directions of the vortices are naturally binary (either clockwise (CW) or counter-clockwise (CCW)), which can be associated with one bit of information, and thus the magnetic vortices have been of great interest recently. A vortex-circulation-based memory requires the perfect controllability of the circulation direction. From the circulation point of view, there are four possible ground states in a nanodisk pair: (CCW, CCW), (CCW, CW), (CW, CCW) and (CW, CW). In a perfect circular nanodisk, CW and CCW states are degenerate because of the high symmetry of the system. However, the circulation of the magnetic vortex is known to be controlled by introducing the asymmetry. It has been reported that the magnetic vortices with opposite (the same) circulations are realized in one-side-flat disk pair. That means in one-side-flat nanodisk pair only the control of two of these four ground states is possible, eg., (CCW, CW), (CW, CCW) or (CCW, CCW), (CW, CW). We found that the reversal of the magnetic vortex circulation is affected by the nanodisk thickness as well. By further introducing another asymmetry, different thickness, the control of the four circulation ground states is achieved in a nanodisk pair. In this work, the controllability of the four ground states in a nanodisk pair was numerically investigated via micromagnetic simulations. The results show that in a single one-side-flat nanodisk, there exists a preferred rotational sense at the remanent state after the nanodisk is saturated by the external magnetic field, applied parallel to the flat edge of the nanodisk. The shape anisotropy is the primary cause of this phenomenon. We further found that the obtained rotational senses of the magnetization in the vortex state in nanodisks with the same geometrical parameters but different thickness (20 nm and 50 nm) are opposite for the same direction of the externally applied field. This is attributed to the competition between the demagnetization field energy and the exchange energy during the vortex formation. The method we proposed provides a simple means of controlling the vortex state that can thus become a useful tool for designing vortex-based devices.

Ultralow-Power Electrically Activated Lab-on-a-Chip Plasmonic Tweezers

We propose ultralow-power plasmonic tweezers with no external optical source. They consist of a one-dimensional array of graphene-based plasmonic units driven by the optical transitions within the underlying array of $(\mathrm{Al},\mathrm{In})\mathrm{As}/(\mathrm{Ga},\mathrm{In})\mathrm{As}/(\mathrm{Al},\mathrm{In})\mathrm{As}/(\mathrm{Ga},\mathrm{In})\mathrm{As}/(\mathrm{Al},\mathrm{In})\mathrm{As}$ quantum cascaded heterostructures (QCHs), electrically biased in series. Each QCH unit formed in a nanopillar can act as a built-in optical source required for exciting the localized surface plasmons (LSPs) at the surface of the overlying circular graphene nanodisk. The stimulated emission due to intersubband transition within each optical source evanesces through the top $(\mathrm{Al},\mathrm{In})\mathrm{As}$ cladding layer and interacts with the overlying graphene nanodisk, inducing the LSPs required for the formation of the plasmonic tweezers. Numerical simulations show, under 145--170 mV applied voltages, that the tweezers with graphene nanodisks of 16--30 nm in diameter and chemical potentials of 0.5--0.7 eV can trap polystyrene nanoparticles of 9 nm in diameter and larger, demonstrating acceptable sensitivities for variations in the nanoparticle diameter and refractive index. These lab-on-a-chip plasmonic tweezers, benefiting from their small footprints and ultralow power consumptions, which are capable of sensing and trapping nanoparticles without requiring expensive external optical sources, open up a different horizon for developing compact on-chip plasmonic tweezers.

Perfect Absorption Efficiency Circular Nanodisk Array Integrated with a Reactive Impedance Surface with High Field Enhancement.

Infrared (IR) absorbers based on a metal–insulator–metal (MIM) have been widely investigated due to their high absorption performance and simple structure. However, MIM absorbers based on ultrathin spacers suffer from low field enhancement. In this study, we propose a new MIM absorber structure to overcome this drawback. The proposed absorber utilizes a reactive impedance surface (RIS) to boost field enhancement without an ultrathin spacer and maintains near-perfect absorption by impedance matching with the vacuum. The RIS is a metallic patch array on a grounded dielectric substrate that can change its surface impedance, unlike conventional metallic reflectors. The final circular nanodisk array mounted on the optimum RIS offers an electric field enhancement factor of 180 with nearly perfect absorption of 98% at 230 THz. The proposed absorber exhibits robust performance even with a change in polarization of the incident wave. The RIS-integrated MIM absorber can be used to enhance the sensitivity of a local surface plasmon resonance (LSPR) sensor and surface-enhanced IR spectroscopy.

Reversing the circulation of ferromagnetic nanodisks with a local circular magnetic field

Ferromagnetic nanodisks have a unique closed-flux vortex state with two degrees of freedom that results in four different magnetization states that are degenerate in energy and stable against thermal fluctuations. Such disks could be interesting for magnetic memory devices if the independent switching of each degree of freedom can be realized. Polarity switching of the vortex core has been demonstrated, but it is difficult to manipulate switching of the vortex due to the high symmetry of the structure. In this work, we reverse the circulation direction of a circular ferromagnetic nanodisk by applying a local circular Oersted field via a metallic atomic force microscope tip placed at the center of the disk. The resulting field reverses the circulation of the vortex without switching the orientation of the core. Switching of the vortex is accomplished by the sudden increase in the current that occurs when there is dielectric breakdown of a thin insulating layer on top of the disk. Micromagnetic simulations indicate that a line current concentrated in the center of a nanodisk can reverse the magnetization of the disk at a value over one order of magnitude smaller than the current required if the current is instead uniformly distributed across the cross section of disk. These results can be applied to reducing the switching current in circularly symmetric device structures.

Influence of Lipid Compositions in the Events of Retinal Schiff Base of Bacteriorhodopsin Embedded in Covalently Circularized Nanodiscs: Thermal Isomerization, Photoisomerization, and Deprotonation.

Covalently circularized nanodiscs using circular membrane scaffold protein (MSP) serve as a suitable membrane mimetic for transmembrane proteins by providing stability and tunability in lipid compositions, providing controllable biological environments for targeted proteins. In this work, monomeric bacteriorhodopsin (mbR) was embedded in lipid nanodiscs of different lipid compositions using negatively charged lipid dioleoyl phosphatidylglycerol (DOPG) and the zwitterion lipid dioleoyl phosphatidylcholine (DOPC), and the events associated with the retinal Schiff base, including the thermal isomerization during the dark adaptation, photoisomerization, and deprotonation, were investigated. The retinal thermal isomerization from all-trans, 15-anti to the 13-cis, 15-syn configuration during the dark adaptation was accelerated in the DOPG bilayer, whereas the processes in the DOPC bilayer and in Triton X-100 micelles were similar. This observation indicated that the negatively charged lipid reduced the barrier for retinal thermal isomerization at C13═C14-C15═N in the ground electronic state. Furthermore, the broader absorption contour of mbR in the DOPC nanodisc probably indicated various retinal isomers in the light-adapted state, consistent with the observed nontwo-state dark adaptation kinetics. Moreover, the kinetics of the photoisomerization of the retinal was slightly decelerated upon increasing the content of DOPC. However, the cascading deprotonation of the protonated Schiff base is not dependent on the types of the surrounding lipids in the nanodiscs. In summary, our research deepens the understanding of the coupling between lipid membrane and the photochemistry of bR retinal Schiff base. Combined with the results of our previous works (Lee, T.-Y.; Yeh, V.; Chuang, J.; Chan, J. C. C.; Chu, L.-K.; Yu, T.-Y. Biophys. J. 2015, 109, 1899-1906; Kao, Y.-M.; Cheng, C.-H.; Syue, M.-L.; Huang, H.-Y.; Chen, I-C.; Yu, T.-Y.; Chu, L.-K. J. Phys. Chem. B 2019, 123, 2032-2039), these outcomes extend our understanding of the control of photochemistry and biophysical events for other photosynthetic proteins via altering the lipid environments.

Colorimetric probe for Ni2+ based on shape transformation of triangular silver nanoprisms upon H2O2 etching

In this article, a highly sensitive and selective colorimetric method for the detection of Ni2+ ions is presented on the basis of hydrogen peroxide etching of triangular silver nanoprisms (AgNPrs). Triangular AgNPrs were synthesized by a modified seed-mediated growth method, and upon addition of Ni2+ ions these triangular AgNPrs showed a significant colorimetric change from blue to reddish brown, and eventually yellow to as a function of the Ni2+ ion concentration. Ni2+ ion do not act as a selective etchant to triangular AgNPrs, but instead serve as a catalyst for the generation of H2O2 in a citrate-capped triangular AgNPr colloidal solution. The oxidative etching with H2O2 formed in the colloidal solution sculptured the sharp corners/edges of the triangular AgNPrs to produce circular Ag nanodisk. Selectivity tests for Ni2+ ions by the triangular AgNPr probe were performed in the presence of other various cations and anions at the optimized conditions (pH 8, 15 °C, and 30 min of reaction time). The probe showed a high linearity between absorption ratio (A475/A750) and concentration in the range of 0 to 30 μM of Ni2+, with a limit of detection of 21.6 nM in aqueous solution. The color and UV–vis absorption spectrum of the triangular AgNPr probe indicated that it is quite stable for two weeks in ambient temperature and pH ranges of 4.0˜10.0. This triangular AgNPr probe was successfully employed for the detection of Ni2+ ions in spiked tap and pond water samples.

Copper Sulfide Nanodisks and Nanoprisms for Photoacoustic Ovarian Tumor Imaging.

Transvaginal ultrasound is widely used for ovarian cancer screening but has a high false positive rate. Photoacoustic imaging provides additional optical contrast to supplement ultrasound and might be able to improve the accuracy of screening. Here, we report two copper sulfide (CuS) nanoparticles types (nanodisks and triangular nanoprisms) as the photoacoustic contrast agents for imaging ovarian cancer. Both CuS nanoprisms and nanodisks were ~6 nm thick and ~26 nm wide and were coated with poly(ethylene glycol) to make them colloidally stable in phosphate buffered saline (PBS) for at least 2 weeks. The CuS nanodisks and nanoprisms revealed strong localized surface plasmon resonances with peak maxima at 1145 nm and 1098 nm, respectively. Both nanoparticles types had strong and stable photoacoustic intensity with detection limits below 120 pM. The circular CuS nanodisk remained in the circulation of nude mice (n=4) and xenograft 2008 ovarian tumors (n=4) 17.9-fold and 1.8-fold more than the triangular nanoprisms, respectively. Finally, the photoacoustic intensity of the tumors from the mice (n=3) treated with CuS nanodisks was 3.0-fold higher than the baseline. The tumors treated with nanodisks had a characteristic peak at 920 nm in the spectrum to potentially differentiate the tumor from adjacent tissues.

Circularized and solubility-enhanced MSPs facilitate simple and high-yield production of stable nanodiscs for studies of membrane proteins in solution.

Recently, an enzymatic reaction was utilized to covalently link the N and C termini of membrane scaffold proteins to produce circularized nanodiscs that were more homogeneous and stable than standard nanodiscs. We continue this development and aim for obtaining high yields of stable and monodisperse nanodiscs for structural studies of membrane proteins by solution small-angle scattering techniques. Based on the template MSP1E3D1, we designed an optimized membrane scaffold protein (His-lsMSP1E3D1) with a sortase recognition motif and high abundance of solubility-enhancing negative charges. With these modifications, we show that high protein expression is maintained and that the circularization reaction is efficient, such that we obtain a high yield of circularized membrane scaffold protein (csMSP1E3D1) and downstream circularized nanodiscs. We characterize the circularized protein and corresponding nanodiscs biophysically by small-angle X-ray scattering, size-exclusion chromatography, circular dichroism spectroscopy, and light scattering and compare to noncircularized samples. First, we show that circularized and noncircularized (lsMSP1E3D1) nanodiscs are structurally similar and have the expected nanodisc structure. Second, we show that lsMSP1E3D1 nanodiscs are more stable compared to the template MSP1E3D1 nanodiscs as an effect of the extra negative charges and that csMSP1E3D1 nanodiscs have further improved stability as an effect of circularization. Finally, we show that a membrane protein can be efficiently incorporated in csMSP1E3D1 nanodiscs. Large-scale production methods for circularized nanodiscs with improved thermal and temporal stability will facilitate better access to the nanodisc technology and enable applications at physiologically relevant temperatures.

Tunable triple-band graphene refractive index sensor with good angle-polarization tolerance

Abstract In this article, we design a triple-band graphene refractive index sensor with good angle-polarization tolerance, which is composed of graphene elliptic–circular nanodisk resonators periodically arranged on a dielectric substrate. The numerical results indicate that the resonant wavelengths of three modes of graphene resonators show consecutively linear tunability when the refractive index of the surrounding medium varies, and the sensitivity is up to 11. 56 μ m /RIU. The sensing range can be further adjusted by controlling the doping level of graphene. In addition, the proposed graphene sensor maintains polarization insensitivity over a wide angular range (0–60°), showing very good angle-polarization tolerance. This work enables us to achieve a tunable triple-band sensor and provides potential value for label-free biomedical sensing.

Double and triple-wavelength plasmonic demultiplexers based on improved circular nanodisk resonators

Plasmonic demultiplexers using improved circular nanodisk resonators (CNRs) and metal-insulator-metal waveguides have been designed. The proposed structures use air and silver as insulator and metal layers, respectively. The relative permittivity of silver has been characterized by Drude, Palik, and Drude–Lorentz models in our finite-difference time-domain simulations. To obtain demultiplexers, first two filters based on improved CNRs are designed. One of the most outstanding features of a CNR is that the resonance wavelength can be tuned by changing its radius. It is shown that increasing the CNRs radii for the single-mode bandpass filter increases the resonance wavelength, linearly. Accordingly, double and triple-wavelength demultiplexers (DeMuxes) have been proposed for the wavelength range of 600 to 2000 nm. Due to their small area, the proposed demultiplexers can be applied in integrated optical circuits for optical communication purposes.

Highly Efficient Transfer of 7TM Membrane Protein from Native Membrane to Covalently Circularized Nanodisc

Incorporating membrane proteins into membrane mimicking systems is an essential process for biophysical studies and structure determination. Monodisperse lipid nanodiscs have been found to be a suitable tool, as they provide a near-native lipid bilayer environment. Recently, a covalently circularized nanodisc (cND) assembled with a membrane scaffold protein (MSP) in circular form, instead of conventional linear form, has emerged. Covalently circularized nanodiscs have been shown to have improved stability, however the optimal strategies for the incorporation of membrane proteins, as well as the physicochemical properties of the membrane protein embedded in the cND, have not been studied. Bacteriorhodopsin (bR) is a seven-transmembrane helix (7TM) membrane protein, and it forms a two dimensional crystal consisting of trimeric bR on the purple membrane of halophilic archea. Here it is reported that the bR trimer in its active form can be directly incorporated into a cND from its native purple membrane. Furthermore, the assembly conditions of the native purple membrane nanodisc (PMND) were optimized to achieve homogeneity and high yield using a high sodium chloride concentration. Additionally, the native PMND was demonstrated to have the ability to assemble over a range of different pHs, suggesting flexibility in the preparation conditions. The native PMND was then found to not only preserve the trimeric structure of bR and most of the native lipids in the PM, but also maintained the photocycle function of bR. This suggests a promising potential for assembling a cND with a 7TM membrane protein, extracted directly from its native membrane environment, while preserving the protein conformation and lipid composition.

A Split-Intein-Based Method for the Efficient Production of Circularized Nanodiscs for Structural Studies of Membrane Proteins.

Phospholipid nanodiscs are a native-like membrane mimetic that is suitable for structural studies of membrane proteins. Although nanodiscs of different sizes exist for various structural applications, their thermal and long-term stability can vary considerably. Covalently circularized nanodiscs are a perfect tool to overcome these limitations. Existing methods for the production of circularized nanodiscs can be time-consuming and technically demanding. Therefore, an easy in vivo approach, in which circularized membrane scaffold proteins (MSPs) can be directly obtained from Escherichia coli culture, is reported herein. Nostoc punctiforme DnaE split-intein fusions with MSPs of various lengths are used and consistently provide circularized nanodiscs in high yields. With this approach, a large variety of circularized nanodiscs, ranging from 7 to 26 nm in diameter, that are suitable for NMR spectroscopy and electron microscopy (EM) applications can be prepared. These nanodiscs are superior to those of the corresponding linear versions in terms of stability and size homogeneity, which affects the quality of NMR spectroscopy data and EM experiments. Due to their long-term stability and homogeneity, the presented small circular nanodiscs are suited for high-resolution NMR spectroscopy studies, as demonstrated with two membrane proteins of 17 or 32 kDa in size. The presented method will provide easy access to circularized nanodiscs for structural studies of membrane proteins and for applications in which a defined and stable nanodisc size is required.