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Self-assembly of purified apolipoproteins and phospholipids results in the formation of nanometer-sized lipoprotein complexes, referred to as nanolipoprotein particles (NLPs). These bilayer constructs are fully soluble in aqueous environments and hold great promise as a model system to aid in solubilizing membrane proteins. Size variability in the self-assembly process has been recognized for some time, yet limited studies have been conducted to examine this phenomenon. Understanding the source of this heterogeneity may lead to methods to mitigate heterogeneity or to control NLP size, which may be important for tailoring NLPs for specific membrane proteins. Here, we have used atomic force microscopy, ion mobility spectrometry, and transmission electron microscopy to quantify NLP size distributions on the single-particle scale, specifically focusing on assemblies with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a recombinant apolipoprotein E variant containing the N-terminal 22 kDa fragment (E422k). Four discrete sizes of E422k/DMPC NLPs were identified by all three techniques, with diameters centered at ∼14.5, 19, 23.5, and 28 nm. Computer simulations suggest that these sizes are related to the structure and number of E422k lipoproteins surrounding the NLPs and particles with an odd number of lipoproteins are consistent with the double-belt model, in which at least one lipoprotein adopts a hairpin structure.

In this paper, we propose active broadband plasmonic switches based on a periodic array of metallic dimers of nanodiscs with progressively increasing diameters on a gold coated SiO 2 substrate with a VO 2 thin film as a spacer between the nanodiscs and the underlying plasmonic substrate. Periodic arrays based on unit cells with plasmonic nanodisc dimers of varying diameters are employed to couple multiple wavelengths of the incident radiation into the plasmonic modes of the nanostructure, which results in a broadband plasmonic response. We employ a vanadium dioxide (VO 2) thin film as a spacer between these periodic sets of plasmonic nanodisc-dimers and the underlying gold coated substrate to achieve broadband switching. On exposure to voltage, infrared light or heat, the VO 2 spacer layer changes from its semiconducting state to its metallic state. This transformation leads to significant changes in its optical properties leading to changes in the reflectance spectra of the proposed nanostructure. This results in an efficient switching action over wide range of wavelengths spanning the C, L and U bands of optical communication. We demonstrate a broadband extinction ratio of 5 dB over an operating wavelength range of 650 nm spanning from 1460 nm to 2110 nm, and a broadband extinction ratio of 4 dB over an operating wavelength range of 702 nm spanning from 1432 nm to 2134 nm. In addition, we demonstrate the trade-off between the extinction ratio and the operating wavelength range of these switches. An exhaustive analysis of these switches has been carried out using finite difference time domain (FDTD) modelling to demonstrate that the spectral range over which switching can be achieved by employing these switches can be varied by varying the geometrical parameters of the proposed switches and thus these switches offer the flexibility of being employed for different wavelength regimes. These switches can be potentially employed in optical communication networks or as switching elements in plasmonic circuits. • Plasmonic nanodiscs with progressively increasing diameters on VO 2 for broadband plasmonic switching. • Broadband extinction ratio of 5 dB over an operating wavelength range of 650 nm. • Trade-off between the extinction ratio and the broadband operating wavelength range. • Tunability of the broadband spectral range by optimization of the geometrical parameters of the switch. [ABSTRACT FROM AUTHOR]

Background: The peptide MS2-L represents toxins of the ssRNA Leviviridae phage family and consists of a predicted N-terminal soluble domain followed by a transmembrane domain. MS2-L mediates bacterial cell lysis through the formation of large lesions in the cell envelope, but further details of this mechanism as a prerequisite for applied bioengineering studies are lacking. The chaperone DnaJ is proposed to modulate MS2-L activity, whereas other cellular targets of MS2-L are unknown. Methods: Here, we provide a combined in vitro and in vivo overexpression approach to reveal molecular insights into MS2-L action and its interaction with DnaJ. Full-length MS2-L and truncated derivatives were synthesized cell-free and co-translationally inserted into nanodiscs or solubilized in detergent micelles. By native liquid bead ion desorption mass spectrometry, we demonstrate that MS2-L assembles into high oligomeric states after membrane insertion. Results: Oligomerization is directed by the transmembrane domain and is impaired in detergent environments. Studies with truncated MS2-L derivatives provide evidence that the soluble domain acts as a modulator of oligomer formation. DnaJ strongly interacts with MS2-L in membranes as well as in detergent environments. However, this interaction affects neither the MS2-L membrane insertion efficiency nor its oligomerization in nanodisc membranes. In accordance with the in vitro data, the assembly of MS2-L derivatives into large membrane located clusters was monitored by overexpression of corresponding fusions with fluorescent monitors in E. coli cells. Analysis by cryo-electron microscopy indicates that lesion formation is initiated in the outer membrane, followed by disruption of the peptidoglycan layer and disintegration of the inner membrane. Conclusion: MS2-L forms oligomeric complexes similar to the related phage toxin ΦX174-E. The oligomeric interface of both peptides is located within their transmembrane domains. We propose a potential function of the higher-order assembly of small phage toxins in membrane disintegration and cell lysis., Competing Interests: All authors declared that there are no conflicts of interest., (© The Author(s) 2023.)

Membrane proteins are a highly relevant class of biological molecules and comprise ∼60% of current drug targets. Before being analyzed by structural, biochemical, and biophysical methods, membrane proteins must first be extracted from cellular membranes - often using detergents. Detergent-extracted membrane proteins are amenable to analysis by structural, biochemical, and biophysical techniques. In certain cases, however, detergents can disturb native protein conformations and/or biological activity. This has led to the development of membrane mimetics, which stabilize membrane proteins in a native membrane-like environment that is water-soluble and detergent-free. This review provides an overview of recent developments in the membrane mimetic field, with a focus on nanodiscs, Saposin lipid nanoparticles (SapNPs), peptidiscs, and SMA lipid particles (SMALPs) - and highlights their utility for supporting biophysical, biochemical, and structural characterization of membrane proteins and complexes., (© 2023 The Author(s).)

Oleanolic acid derivative DKS26 has hypolipidemic, islet, and hepatoprotective effects. However, high lipophilicity and low water solubility led to DKS26 extremely low oral bioavailability. Herein, lipid-based nanocarriers, including lipid nanodiscs (sND/DKS26) and liposomes (sLip/DKS26), are prepared to improve DKS26 oral absorption. In comparison to free DKS26 (5.81%), the absolute oral bioavailabilities are significantly increased to 29.47% (sND/DKS26) and 37.25% (sLip/DKS26) without detectable toxicity or immunogenicity even after repeated administrations. Both sND/DKS26 and sLip/DKS26 significantly reduce the feeding glucose level and the AUC of OGTT in db/db diabetic mice. Aiding by the newly developed scFv-based nanocarrier separation methods, no intact nanocarriers are detected in blood circulation after oral administration, suggesting that both formulations are unable to penetrate the intestinal epithelium. They enhance DKS26 absorption mainly by improving intestinal cell uptake and rapid intracellular release of the payload. Since pre-existing anti-PEG is widely detected in humans, the present oral absorption pathway of both nanocarriers successfully avoids unfavorable immunological responses after interaction with anti-PEG antibodies. The application of lipid-based nanocarriers paves an efficient and safe avenue for the clinical translation and application of poorly soluble therapeutics derived from traditional Chinese medicine., (© 2023 Wiley-VCH GmbH.)

The angiotensin-converting enzyme 2 (ACE2), as a functional receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is essential for assessing potential hosts and treatments. However, many studies are based on its truncated version but not full-length structure. Indeed, a single transmembrane (TM) helix presents in the full-length ACE2, influencing its interaction with SARS-CoV-2. Therefore, synthesis of the full-length ACE2 is an urgent requirement. Here, cell-free membrane protein synthesis systems (CFMPSs) are constructed for full-length membrane proteins. MscL is screened as a model among ten membrane proteins based on their expression and solubility. Next, CFMPSs are constructed and optimized based on natural vesicles, vesicles with four membrane proteins removed or two chaperonins added, and 37 types of nanodiscs. They all increase membrane protein solubility to over 50%. Finally, the full-length ACE2 of 21 species are successfully expressed with yields between 0.4 and 0.9 mg mL -1 . The definite functional differences from the truncated version suggest that the TM region affects ACE2's structure and function. CFMPSs can be extended to more membrane proteins, paving the way for further applications., (© 2023 Wiley-VCH GmbH.)

The antiapoptotic protein Bcl-xL is a major regulator of cell death and survival, but many aspects of its functions remain elusive. It is mostly localized in the mitochondrial outer membrane (MOM) owing to its C-terminal hydrophobic α-helix. In order to gain further information about its membrane organization, we set up a model system combining cell-free protein synthesis and nanodisc insertion. We found that, contrary to its proapoptotic partner Bax, neosynthesized Bcl-xL was spontaneously inserted into nanodiscs. The deletion of the C-terminal α-helix of Bcl-xL prevented nanodisc insertion. We also found that nanodisc insertion protected Bcl-xL against the proteolysis of the 13 C-terminal residues that occurs during expression of Bcl-xL as a soluble protein in E. coli . Interestingly, we observed that Bcl-xL increased the insertion of Bax into nanodiscs, in a similar way to that which occurs in mitochondria. Cell-free synthesis in the presence of nanodiscs is, thus, a suitable model system to study the molecular aspects of the interaction between Bcl-xL and Bax during their membrane insertion.

The active transport of ions across biological membranes requires their hydration shell to interact with the interior of membrane proteins. However, the influence of the external lipid phase on internal dielectric dynamics is hard to access by experiment. Using the octahelical transmembrane architecture of the copper-transporting P1B-type ATPase from Legionella pneumophila as a model structure, we have established the site-specific labeling of internal cysteines with a polarity-sensitive fluorophore. This enabled dipolar relaxation studies in a solubilized form of the protein and in its lipid-embedded state in nanodiscs. Time-dependent fluorescence shifts revealed the site-specific hydration and dipole mobility around the conserved ion-binding motif. The spatial distribution of both features is shaped significantly and independently of each other by membrane lateral pressure. [ABSTRACT FROM AUTHOR]

A promising tool in membrane research is the use of the styrene-maleic acid (SMA) copolymer to solubilize membranes in the form of nanodiscs. Since membranes are heterogeneous in composition, it is important to know whether SMA thereby has a preference for solubilization of either specific types of lipids or specific bilayer phases. Here, we investigated this by performing partial solubilization of model membranes and analyzing the lipid composition of the solubilized fraction. We found that SMA displays no significant lipid preference in homogeneous binary lipid mixtures in the fluid phase, even when using lipids that by themselves show very different solubilization kinetics. By contrast, in heterogeneous phase-separated bilayers, SMA was found to have a strong preference for solubilization of lipids in the fluid phase as compared to those in either a gel phase or a liquid-ordered phase. Together the results suggest that (1) SMA is a reliable tool to characterize native interactions between membrane constituents, (2) any solubilization preference of SMA is not due to properties of individual lipids but rather due to properties of the membrane or membrane domains in which these lipids reside and (3) exploiting SMA resistance rather than detergent resistance may be an attractive approach for the isolation of ordered domains from biological membranes. [ABSTRACT FROM AUTHOR]

TonB-dependent transporters are β-barrel outer membrane proteins occluded by a plug domain. Upon ligand binding, these transporters extend a periplasmic motif termed the TonB box. The TonB box permits the recruitment of the inner membrane protein complex TonB-ExbB-ExbD, which drives import of ligands in the cell periplasm. It is unknown precisely how the plug domain is moved aside during transport nor have the intermediate states between TonB recruitment and plug domain movement been characterized biochemically. Here we employ nanodiscs, native gel electrophoresis, and scintillation proximity assays to determine the binding kinetics of vitamin B 12 to BtuB. The results show that ligand-bound BtuB recruits a monomer of TonB (TonB ∆1-31 ), which in turn increases retention of vitamin B 12 within the transporter. The TonB box and the extracellular residue valine 90 that forms part of the vitamin B 12 binding site are essential for this event. These results identify a novel step in the TonB-dependent transport process. They show that TonB binding to BtuB trap the ligand, possibly until the ExbB-ExbD complex is activated or recruited to ensure subsequent transport. [ABSTRACT FROM AUTHOR]

The choice of a suitable membrane mimicking environment is of fundamental importance for the characterization of structure and function of membrane proteins. In this respect, usage of the lipid bilayer nanodisc technology provides a unique potential for nuclear magnetic resonance (NMR)-based studies. This review summarizes the recent advances in this field, focusing on (i) the strengths of the system, (ii) the bottlenecks that may be faced, and (iii) promising capabilities that may be explored in future studies. [ABSTRACT FROM AUTHOR]

Over the past 50 years there has been considerable progress in our understanding of biomolecular interactions at an atomic level. This in turn has allowed molecular simulation methods employing full atomistic modelling at ever larger scales to develop. However, some challenging areas still remain where there is either a lack of atomic resolution structures or where the simulation system is inherently complex. An area where both challenges are present is that of membranes containing membrane proteins. In this review we analyse a new practical approach to membrane protein study that offers a potential new route to high resolution structures and the possibility to simplify simulations. These new approaches collectively recognise that preservation of the interaction between the membrane protein and the lipid bilayer is often essential to maintain structure and function. The new methods preserve these interactions by producing nano-scale disc shaped particles that include bilayer and the chosen protein. Currently two approaches lead in this area: the MSP system that relies on peptides to stabilise the discs, and SMALPs where an amphipathic styrene maleic acid copolymer is used. Both methods greatly enable protein production and hence have the potential to accelerate atomic resolution structure determination as well as providing a simplified format for simulations of membrane protein dynamics. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. [ABSTRACT FROM AUTHOR]

Arabidopsis thaliana Regulator of G protein Signalling 1 (AtRGS1) is a protein with a predicted N-terminal 7-transmembrane (7TM) domain and a C-terminal cytosolic RGS1 box domain. The RGS1 box domain exerts GTPase activation (GAP) activity on Gα (AtGPA1), a component of heterotrimeric G protein signaling in plants. AtRGS1 may perceive an exogenous agonist to regulate the steady-state levels of the active form of AtGPA1. It is uncertain if the full-length AtRGS1 protein exerts any atypical effects on Gα, nor has it been established exactly how AtRGS1 contributes to perception of an extracellular signal and transmits this response to a G-protein dependent signaling cascade. Further studies on full-length AtRGS1 have been inhibited due to the extreme low abundance of the endogenous AtRGS1 protein in plants and lack of a suitable heterologous system to express AtRGS1. Here, we describe methods to produce full-length AtRGS1 by cell-free synthesis into unilamellar liposomes and nanodiscs. The cell-free synthesized AtRGS1 exhibits GTPase activating activity on Gα and can be purified to a level suitable for biochemical analyses. [ABSTRACT FROM AUTHOR]

During the last decade, and with increasing intensity, the potential for using reconstituted high-density lipoprotein (rHDL) particles to deliver hydrophobic drugs to impaired cells and tissues has been explored. Here, we evaluate various parameters that should be considered when utilizing discoidal rHDL particles as a drug delivery platform. Key parameters such as preparation basics, pronounced statistical variation in drug incorporation across rHDL particles, effects of lipid composition on HDL/rHDL in vivo and vitro dynamics/particle stability, and pharmacokinetic/safety data from rHDL infusion studies in human subjects will be addressed including the innate receptors and native functions of HDL. The broad but detailed information presented in this work could also be deployed in other rHDL-related research. However, the major aim of this review is to point out factors that have the potential to advance rHDL research toward realizing the ‘magic bullet’ for lipophilic and hydrophilic drug delivery in various clinical contexts. [ABSTRACT FROM AUTHOR]

Dynamics are thought to be important features of structure and signaling in the cytoplasmic domain of bacterial chemoreceptors. However, little is known about which structural features are dynamic. For this largely helical domain, comprising a four-helix bundle and an extended four-helix coiled coil, functionally important structural dynamics likely involves helical mobility and stability. To investigate, we used continuous wave EPR spectroscopy and site-specific spin labels that directly probed, in essentially physiological conditions, the mobility of helical backbones in the cytoplasmic domain of intact chemoreceptor Tar homodimers inserted into lipid bilayers of Nanodiscs. We observed differences among functional regions, between companion helices in helical hairpins of the coiled coil and between receptor conformational states generated by adaptational modification. Increased adaptational modification decreased helical dynamics while preserving dynamics differences among functional regions and between companion helices. In contrast, receptor ligand occupancy did not have a discernable effect on dynamics to which our approach was sensitive, implying that the two sensory inputs alter different chemoreceptor features. Spectral fitting indicated that differences in helical dynamics we observed for ensemble spin-label mobility reflected differences in proportions of a minority receptor population in which the otherwise helical backbone was essentially disordered. We suggest that our measurements provided site-specific snapshots of equilibria between a majority state of well-ordered helix and a minority state of locally disordered polypeptide backbone. Thus, the proportion of polypeptide chain that is locally and presumably transiently disordered is a structural feature of cytoplasmic domain dynamics that varies with functional region and modification-induced signaling state. [ABSTRACT FROM AUTHOR]

Cytochrome P450 17A1 (CYP17A1) is an important drug target for castration resistant prostate cancer. It is a bi-functional enzyme, catalyzing production of glucocorticoid precursors by hydroxylation of pregnene-nucleus, and androgen biosynthesis by a second C C lyase step, at the expense of glucocorticoid production. Cytochrome b 5 (cyt b 5 ) is known to be a key regulator of the androgen synthesis reaction in vivo , by a mechanism that is not well understood. Two hypotheses have been proposed for the mechanism by which cyt b 5 increases androgen biosynthesis. Cyt b 5 could act as an allosteric effector, binding to CYP17A1 and either changing its selective substrate affinity or altering the conformation of the P450 to increase the catalytic rate or decrease unproductive uncoupling channels. Alternatively, cyt b 5 could act as a redox donor for supply of the second electron in the P450 cycle, reducing the oxyferrous complex to form the reactive peroxo-intermediate. To understand the mechanism of lyase enhancement by cyt b 5 , we generated a redox-inactive form of cyt b 5 , in which the heme is replaced with a Manganese-protoporphyrin IX (Mn- b 5 ), and investigated enhancement of androgen producing lyase reaction by CYP17A1. Given the critical significance of a stable membrane anchor for all of the proteins involved and the need for controlled stoichiometric ratios, we employed the Nanodisc system for this study. The redox inactive form was observed to have no effect on the lyase reaction, while reactions with the normal heme-iron containing cyt b 5 were enhanced ∼5 fold as compared to reactions in the absence of cyt b 5 . We also performed resonance Raman measurements on ferric CYP17A1 bound to Mn- b 5 . Upon addition of Mn- b 5 to Nanodisc reconstituted CYP17A1, we observed clear evidence for the formation of a b 5 -CYP17A1 complex, as noted by changes in the porphyrin modes and alteration in the proximal Fe S vibrational frequency. Thus, although Mn- b 5 binds to CYP17A1, it is unable to enhance the lyase reaction, strongly suggesting that cyt b 5 has a redox effector role in enhancement of the CYP17A1 mediated lyase reaction necessary for androgen synthesis. [ABSTRACT FROM AUTHOR]

One of the most important classes of proteins in terms of drug targets is cell surface membrane proteins, and yet it is a challenging set of proteins for generating high-quality affinity reagents. In this review, we focus on the use of phage libraries, which display antibody fragments, for generating recombinant antibodies to membrane proteins. Such affinity reagents generally have high specificity and affinity for their targets. They have been used for cell staining, for promoting protein crystallization to solve three-dimensional structures, for diagnostics, and for treating diseases as therapeutics. We cover publications on this topic from the past 10 years, with a focus on the various formats of membrane proteins for affinity selection and the diverse affinity selection strategies used. Lastly, we discuss the challenges faced in this field and provide possible directions for future efforts. [ABSTRACT FROM AUTHOR]

Membrane proteins represent one of the most important targets for pharmaceutical companies. Unfortunately, technical limitations have long been a major hindrance in our understanding of the function and structure of such proteins. Recent years have seen the refinement of classical approaches and the emergence of new technologies that have resulted in a significant step forward in the field of membrane protein research. This review summarizes some of the current techniques used for studying membrane proteins, with overall advantages and drawbacks for each method. [ABSTRACT FROM AUTHOR]

Cytochrome P450 CYP3A4 is the main drug-metabolizing enzyme in the human liver, being responsible for oxidation of 50% of all pharmaceuticals metabolized by human P450 enzymes. Possessing a large substrate binding pocket, it can simultaneously bind several substrate molecules and often exhibits a complex pattern of drug–drug interactions. In order to better understand structural and functional aspects of binding of multiple substrate molecules to CYP3A4 we used resonance Raman and UV–VIS spectroscopy to document the effects of binding of synthetic testosterone dimers of different configurations, cis -TST 2 and trans -TST 2 . We directly demonstrate that the binding of two steroid molecules, which can assume multiple possible configurations inside the substrate binding pocket of monomeric CYP3A4, can lead to active site structural changes that affect functional properties. Using resonance Raman spectroscopy, we have documented perturbations in the ferric and Fe-CO states by these substrates, and compared these results with effects caused by binding of monomeric TST. While the binding of trans -TST 2 yields results similar to those obtained with monomeric TST, the binding of cis -TST 2 is much tighter and results in significantly more pronounced conformational changes of the porphyrin side chains and Fe-CO unit. In addition, binding of an additional monomeric TST molecule in the remote allosteric site significantly improves binding affinity and the overall spin shift for CYP3A4 with trans -TST 2 dimer bound inside the substrate binding pocket. This result provides the first direct evidence for an allosteric effect of the peripheral binding site at the protein-membrane interface on the functional properties of CYP3A4. [ABSTRACT FROM AUTHOR]

The NADPH-dependent cytochrome P450 oxidoreductase (POR) is the obligate electron donor to eukaryotic microsomal cytochromes P450 enzymes. The number of PORs within plant species is limited to one to four isoforms, with the most common being two PORs per plant. These enzymes provide electrons to a huge number of different cytochromes P450s (from 50 to several hundred within one plant). Within the eudicotyledons, PORs can be divided into two major clades, POR 1 and POR 2. Based on our own sequencing analysis and publicly available data, we have identified 45 PORs from the angiosperm order Apiales. These were subjected to a phylogenetic analysis along with 237 other publicly available (NCBI and oneKP) POR sequences found within the clade Asterids. Here, we show that the order Apiales only harbor members of the POR 2 clade, which are further divided into two distinct subclades. This is in contrast to most other eudicotyledon orders that have both POR 1 and POR 2. This suggests that through gene duplications and one gene deletion, Apiales only contain members of the POR 2 clade. Three POR 2 isoforms from Thapsia garganica L., Apiaceae, were all full-length in an Illumina root transcriptome dataset (available from the SRA at NCBI). All three genes were shown to be functional upon reconstitution into nanodiscs, confirming that none of the isoforms are pseudogenes. [ABSTRACT FROM AUTHOR]

A room temperature reduction-hydrolysis of Fe(III) precursors such as FeCl3 or Fe(acac)3 in various lyotropic liquid crystal phases (lamellar, hexagonal columnar, or micellar) formed by a range of ionic or neutral surfactants in H2O is shown to be an effective and mild approach for the preparation of iron oxide (IO) nanomaterials with several morphologies (shapes and dimensions), such as extended thin nanosheets with lateral dimensions of several hundred nanometers as well as smaller nanoflakes and nanodiscs in the tens of nanometers size regime. We will discuss the role of the used surfactants and lyotropic liquid crystal phases as well as the shape and size differences depending upon when and how the resulting nanomaterials were isolated from the reaction mixture. The presented synthetic methodology using lyotropic liquid crystal solvents should be widely applicable to several other transition metal oxides for which the described reduction-hydrolysis reaction sequence is a suitable pathway to obtain nanoscale particles.

Bax is a major player in the mitochondrial pathway of apoptosis, by making the Outer Mitochondrial Membrane (OMM) permeable to various apoptogenic factors, including cytochrome c. In order to get further insight into the structure and function of Bax when it is inserted in the OMM, we attempted to reconstitute Bax in nanodiscs. Cell-free protein synthesis in the presence of nanodiscs did not yield Bax-containing nanodiscs, but it provided a simple way to purify full-length Bax without any tag. Purified wild-type Bax (BaxWT) and a constitutively active mutant (BaxP168A) displayed biochemical properties that were in line with previous characterizations following their expression in yeast and human cells followed by their reconstitution into liposomes. Both Bax variants were then reconstituted in nanodiscs. Size exclusion chromatography, dynamic light scattering and transmission electron microscopy showed that nanodiscs formed with BaxP168A were larger than nanodiscs formed with BaxWT. This was consistent with the hypothesis that BaxP168A was reconstituted in nanodiscs as an active oligomer. [Display omitted] • Cell-free synthesis of Bax in the presence of nanodiscs did not allow a direct insertion of the protein • However, Bax could be adequatly purified for biochemical studies • Both BaxWT and the active mutant BaxP168A could be further reconstituted into nanodiscs by co-formation • Nanodiscs containing BaxP168A were larger, suggesting that it was reconstituted as an oligomer [ABSTRACT FROM AUTHOR]

[Display omitted] • Nanodiscs are lipid-based membrane mimetics for structural studies with NMR. • Improved homogeneity and size stability of nanodiscs by MSP circularization. • Higher thermal stability but similar lipid fluidity in cMSP nanodiscs. • Enhanced NMR spectral quality of a membrane protein in cMSP nanodiscs. • cMSP nanodiscs facilitate NMR studies of challenging 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. [ABSTRACT FROM AUTHOR]

Transition metal dichalcogenides (TMDC) possess a unique combination of properties well-suited for the creation of novel optoelectronic and photonic devices. The titanium disulphide (TiS 2) outstands among those as the lightest of TMDCs besides allowing low production cost and high stability. In this work, we present a comprehensive study of electron-phonon interaction induced vibrational properties of suspended TiS 2 hexagonal nanodisks for an extended and elevated temperature range from 88 K to 538 K and a variety of laser powers. Our study reveals a stronger temperature dependence for out-of-plane vibrational mode (A 1g) in comparison to that of in-plane vibration mode (E g) which is attributed to a significant difference between strong intralayer covalent bonds and weak interlayer van der Waals bonds within the material. Furthermore, we observed that both E g and A 1g modes linearly undergo a red shift at higher temperatures, and in addition, a broadening at higher in-laser powers. On the basis of the present study, we propose that the thermal conductivity of the material is 5.049 W/mK. [ABSTRACT FROM AUTHOR]