Search Results (17)

Fluorescent labeling of membrane proteins is essential for exploring their functions, signaling pathways, interaction partners, and structural dynamics. Organic fluorophores are commonly used for this purpose due to their favorable photophysical properties and photostability. However, a persistent challenge is the inaccessibility of the surface-exposed cysteine residues required for site-specific labeling, as these residues often become sequestered within detergent micelles during protein extraction. To address this limitation, we developed an approach based on polymer-encapsulated nanodiscs that preserves the protein’s native-like lipid-bilayer environment while ensuring the accessibility of surface-exposed cysteine residues. In this method, His-tagged proteins embedded in native nanodiscs are retained on a nickel affinity column, allowing for simultaneous purification and labeling by adding fluorescent dyes. This versatile technique was demonstrated with two challenging-to-label membrane proteins, the potassium channel KvAP and the urea channel HpUreI, for which detergent-based labeling had failed. This opens new possibilities for studying a wide range of fluorescently labeled membrane proteins in near-native states, advancing applications in biophysics, structural biology, and drug discovery. Full article

The production of functionally active membrane proteins (MPs) in an adequate membrane environment is a key step in structural biology. Polymer–lipid particles based on styrene and maleic acid (SMA) represent a promising type of membrane mimic, as they can extract properly folded MPs directly from their native lipid environment. However, the original SMA polymer is sensitive to acidic pH levels, which has led to the development of several modifications: SMA-EA, SMA-QA, and others. Here, we introduce a novel SMA derivative with a negatively charged taurine moiety, SMA-tau, and investigate the formation and characteristics of lipid–SMA-EA and lipid–SMA-tau membrane-mimicking particles. Our findings demonstrate that both polymers can form nanodiscs with a patch of lipid bilayer that can undergo phase transitions at temperatures close to those of the lipid bilayer membranes. Finally, we discuss the potential applications of these SMAs for NMR spectroscopy. Full article

Natural rubber (NR) is utilized in more than 40,000 products, and the demand for NR is projected to reach $68.5 billion by 2026. The primary commercial source of NR is the latex of Hevea brasiliensis. NR is produced by the sequential cis-condensation of isopentenyl diphosphate (IPP) through a complex known as the rubber transferase (RTase) complex. This complex is associated with rubber particles, specialized organelles for NR synthesis. Despite numerous attempts to isolate, characterize, and study the RTase complex, definitive results have not yet been achieved. This review proposes an innovative approach to overcome this longstanding challenge. The suggested method involves isolating the RTase complex without using detergents, instead utilizing the native membrane lipids, referred to as “natural nanodiscs”, and subsequently reconstituting the complex on liposomes. Additionally, we recommend the adaptation of large nanodiscs for the incorporation and reconstitution of the RTase complex, whether it is in vitro transcribed or present within the natural nanodiscs. These techniques show promise as a viable solution to the current obstacles. Based on our experimental experience and insights from published literature, we believe these refined methodologies can significantly enhance our understanding of the RTase complex and its role in in vitro NR synthesis. Full article

Nanodiscs belong to a category of water-soluble lipid bilayer nanoparticles. In vivo nanodisc platforms are useful for studying isolated membrane proteins in their native lipid environment. Thus, the development of a practical method for nanodisc reconstruction has garnered consider-able research interest. This paper reports the self-assembly of a mixture of bio-derived cyclic peptide, surfactin (SF), and l-α-dimyristoylphosphatidylcholine (DMPC). We found that SF induced the solubilization of DMPC multilamellar vesicles to form their nanodiscs, which was confirmed by size-exclusion chromatography, dynamic light scattering, and transmission electron microscopy analyses. Owing to its amphiphilic nature, the self-assembled structure prevents the exposure of the hydrophobic lipid core to aqueous media, thus embedding ubiquinol (CoQ10) as a hydrophobic model compound within the inner region of the nanodiscs. These results highlight the feasibility of preparing nanodiscs without the need for laborious procedures, thereby showcasing their potential to serve as promising carriers for membrane proteins and various organic compounds. Additionally, the regulated self-assembly of the DMPC/SF mixture led to the formation of fibrous architectures. These results show the potential of this mixture to function as a nanoscale membrane surface for investigating molecular recognition events. Full article

KCNE3 is a single-pass integral membrane protein that regulates numerous voltage-gated potassium channel functions such as KCNQ1. Previous solution NMR studies suggested a moderate degree of curved α-helical structure in the transmembrane domain (TMD) of KCNE3 in lyso-myristoylphosphatidylcholine (LMPC) micelles and isotropic bicelles with the residues T71, S74 and G78 situated along the concave face of the curved helix. During the interaction of KCNE3 and KCNQ1, KCNE3 pushes its transmembrane domain against KCNQ1 to lock the voltage sensor in its depolarized conformation. A cryo-EM study of KCNE3 complexed with KCNQ1 in nanodiscs suggested a deviation of the KCNE3 structure from its independent structure in isotropic bicelles. Despite the biological significance of KCNE3 TMD, the conformational properties of KCNE3 are poorly understood. Here, all atom molecular dynamics (MD) simulations were utilized to investigate the conformational dynamics of the transmembrane domain of KCNE3 in a lipid bilayer containing a mixture of POPC and POPG lipids (3:1). Further, the effect of the interaction impairing mutations (V72A, I76A and F68A) on the conformational properties of the KCNE3 TMD in lipid bilayers was investigated. Our MD simulation results suggest that the KCNE3 TMD adopts a nearly linear α helical structural conformation in POPC-POPG lipid bilayers. Additionally, the results showed no significant change in the nearly linear α-helical conformation of KCNE3 TMD in the presence of interaction impairing mutations within the sampled time frame. The KCNE3 TMD is more stable with lower flexibility in comparison to the N-terminal and C-terminal of KCNE3 in lipid bilayers. The overall conformational flexibility of KCNE3 also varies in the presence of the interaction-impairing mutations. The MD simulation data further suggest that the membrane bilayer width is similar for wild-type KCNE3 and KCNE3 containing mutations. The Z-distance measurement data revealed that the TMD residue site A69 is close to the lipid bilayer center, and residue sites S57 and S82 are close to the surfaces of the lipid bilayer membrane for wild-type KCNE3 and KCNE3 containing interaction-impairing mutations. These results agree with earlier KCNE3 biophysical studies. The results of these MD simulations will provide complementary data to the experimental outcomes of KCNE3 to help understand its conformational dynamic properties in a more native lipid bilayer environment. Full article

Eight hundred and twenty-six human G protein-coupled receptors (GPCRs) mediate the actions of two-thirds of the human hormones and neurotransmitters and over one-third of clinically used drugs. Studying the structure and dynamics of human GPCRs in lipid bilayer environments resembling the native cell membrane milieu is of great interest as a basis for understanding structure–function relationships and thus benefits continued drug development. Here, we incorporate the human A_2A adenosine receptor (A_2AAR) into lipid nanodiscs, which represent a detergent-free environment for structural studies using nuclear magnetic resonance (NMR) in solution. The [¹⁵N,¹H]-TROSY correlation spectra confirmed that the complex of [u-¹⁵N, ~70% ²H]-A_2AAR with an inverse agonist adopts its global fold in lipid nanodiscs in solution at physiological temperature. The global assessment led to two observations of practical interest. First, A_2AAR in nanodiscs can be stored for at least one month at 4 °C in an aqueous solvent. Second, LMNG/CHS micelles are a very close mimic of the environment of A_2AAR in nanodiscs. The NMR signal of five individually assigned tryptophan indole ¹⁵N–¹H moieties located in different regions of the receptor structure further enabled a detailed assessment of the impact of nanodiscs and LMNG/CHS micelles on the local structure and dynamics of A_2AAR. As expected, the largest effects were observed near the lipid–water interface along the intra- and extracellular surfaces, indicating possible roles of tryptophan side chains in stabilizing GPCRs in lipid bilayer membranes. Full article

Chemokine receptors are extensively involved in a broad range of physiological and pathological processes, making them attractive drug targets. However, despite considerable efforts, there are very few approved drugs targeting this class of seven transmembrane domain receptors to date. In recent years, the importance of including binding kinetics in drug discovery campaigns was emphasized. Therefore, kinetic insight into chemokine–chemokine receptor interactions could help to address this issue. Moreover, it could additionally deepen our understanding of the selectivity and promiscuity of the chemokine–chemokine receptor network. Here, we describe the application, optimization and validation of a homogenous Scintillation Proximity Assay (SPA) for real-time kinetic profiling of chemokine–chemokine receptor interactions on the example of ACKR3 and CXCL12. The principle of the SPA is the detection of radioligand binding to receptors reconstituted into nanodiscs by scintillation light. No receptor modifications are required. The nanodiscs provide a native-like environment for receptors and allow for full control over bilayer composition and size. The continuous assay format enables the monitoring of binding reactions in real-time, and directly accounts for non-specific binding and potential artefacts. Minor adaptations additionally facilitate the determination of equilibrium binding metrics, making the assay a versatile tool for the study of receptor–ligand interactions. Full article

Integral membrane proteins (IMPs) fulfill important physiological functions by providing cell–environment, cell–cell and virus–host communication; nutrients intake; export of toxic compounds out of cells; and more. However, some IMPs have obliterated functions due to polypeptide mutations, modifications in membrane properties and/or other environmental factors—resulting in damaged binding to ligands and the adoption of non-physiological conformations that prevent the protein from returning to its physiological state. Thus, elucidating IMPs’ mechanisms of function and malfunction at the molecular level is important for enhancing our understanding of cell and organism physiology. This understanding also helps pharmaceutical developments for restoring or inhibiting protein activity. To this end, in vitro studies provide invaluable information about IMPs’ structure and the relation between structural dynamics and function. Typically, these studies are conducted on transferred from native membranes to membrane-mimicking nano-platforms (membrane mimetics) purified IMPs. Here, we review the most widely used membrane mimetics in structural and functional studies of IMPs. These membrane mimetics are detergents, liposomes, bicelles, nanodiscs/Lipodisqs, amphipols, and lipidic cubic phases. We also discuss the protocols for IMPs reconstitution in membrane mimetics as well as the applicability of these membrane mimetic-IMP complexes in studies via a variety of biochemical, biophysical, and structural biology techniques. Full article

The succinate-acetate permease (SatP) is an anion channel with six transmembrane domains. It forms different oligomers, especially hexamers in the detergent as well as in the membrane. Solid-state NMR studies of SatP were carried out successfully on SatP complexes by reconstructing the protein into liposomes or retaining the protein in the native membrane of E. coli., where it was expressed. The comparison of ¹³C-¹³C 2D correlation spectra between the two samples showed great similarity, opening the possibility to further study the acetate transport mechanism of SatP in its native membrane environment. Solid-state NMR studies also revealed small chemical shift differences of SatP in the two different membrane systems, indicating the importance of the lipid environment in determining the membrane protein structures and dynamics. Combining different 2D SSNMR spectra, chemical shift assignments were made on some sites, consistent with the helical structures in the transmembrane domains. In the end, we pointed out the limitation in the sensitivity for membrane proteins with such a size, and also indicated possible ways to overcome it. Full article

Membrane proteins work within asymmetric bilayers of lipid molecules that are critical for their biological structures, dynamics and interactions. These properties are lost when detergents dislodge lipids, ligands and subunits, but are maintained in native nanodiscs formed using styrene maleic acid (SMA) and diisobutylene maleic acid (DIBMA) copolymers. These amphipathic polymers allow extraction of multicomponent complexes of post-translationally modified membrane-bound proteins directly from organ homogenates or membranes from diverse types of cells and organelles. Here, we review the structures and mechanisms of transmembrane targets and their interactions with lipids including phosphoinositides (PIs), as resolved using nanodisc systems and methods including cryo-electron microscopy (cryo-EM) and X-ray diffraction (XRD). We focus on therapeutic targets including several G protein-coupled receptors (GPCRs), as well as ion channels and transporters that are driving the development of next-generation native nanodiscs. The design of new synthetic polymers and complementary biophysical tools bodes well for the future of drug discovery and structural biology of native membrane:protein assemblies (memteins). Full article

Membrane proteins evolved to reside in the hydrophobic lipid bilayers of cellular membranes. Therefore, membrane proteins bridge the different aqueous compartments separated by the membrane, and furthermore, dynamically interact with their surrounding lipid environment. The latter not only stabilizes membrane proteins, but directly impacts their folding, structure and function. In order to be characterized with biophysical and structural biological methods, membrane proteins are typically extracted and subsequently purified from their native lipid environment. This approach requires that lipid membranes are replaced by suitable surrogates, which ideally closely mimic the native bilayer, in order to maintain the membrane proteins structural and functional integrity. In this review, we survey the currently available membrane mimetic environments ranging from detergent micelles to bicelles, nanodiscs, lipidic-cubic phase (LCP), liposomes, and polymersomes. We discuss their respective advantages and disadvantages as well as their suitability for downstream biophysical and structural characterization. Finally, we take a look at ongoing methodological developments, which aim for direct in-situ characterization of membrane proteins within native membranes instead of relying on membrane mimetics. Full article

Membrane proteins play a crucial role in cell physiology by participating in a variety of essential processes such as transport, signal transduction and cell communication. Hence, understanding their structure–function relationship is vital for the improvement of therapeutic treatments. Over the last decade, based on the development of detergents, amphipoles and styrene maleic-acid lipid particles (SMALPs), remarkable accomplishments have been made in the field of membrane protein structural biology. Nevertheless, there are still many drawbacks associated with protein–detergent complexes, depending on the protein in study or experimental application. Recently, newly developed membrane mimetic systems have become very popular for allowing a structural and functional characterisation of membrane proteins in vitro. The nanodisc technology is one such valuable tool, which provides a more native-like membrane environment than detergent micelles or liposomes. In addition, it is also compatible with many biophysical and biochemical methods. Here we describe the use of in situ dynamic light scattering to accurately and rapidly probe membrane proteins’ reconstitution into nanodiscs. The adenosine type 2A receptor (A_2AR) was used as a case study. Full article

Nanolipoprotein particles (NLPs), also called “nanodiscs”, are discoidal particles with a patch of lipid bilayer corralled by apolipoproteins. NLPs have long been of interest due to both their utility as membrane-model systems into which membrane proteins can be inserted and solubilized and their physiological role in lipid and cholesterol transport via high-density lipoprotein (HDL) and low-density lipoprotein (LDL) maturation, which are important for human health. Serial femtosecond crystallography (SFX) at X-ray free electron lasers (XFELs) is a powerful approach for structural biology of membrane proteins, which are traditionally difficult to crystallize as large single crystals capable of producing high-quality diffraction suitable for structure determination. To facilitate understanding of the specific role of two apolipoprotein/lipid complexes, ApoA1 and ApoE4, in lipid binding and HDL/LDL particle maturation dynamics, and to develop new SFX methods involving NLP membrane protein encapsulation, we have prepared and crystallized homogeneous populations of ApoA1 and ApoE4 NLPs. Crystallization of empty NLPs yields semi-ordered objects that appear crystalline and give highly anisotropic and diffuse X-ray diffraction, similar to fiber diffraction. Several unit cell parameters were approximately determined for both NLPs from these measurements. Thus, low-background, sample conservative methods of delivery are critical. Here we implemented a fixed target sample delivery scheme utilizing the Roadrunner fast-scanning system and ultra-thin polymer/graphene support films, providing a low-volume, low-background approach to membrane protein SFX. This study represents initial steps in obtaining structural information for ApoA1 and ApoE4 NLPs and developing this system as a supporting scaffold for future structural studies of membrane proteins crystalized in a native lipid environment. Full article

During the late phase of the HIV-1 replication cycle, the Gag polyproteins are transported to the plasma membrane (PM) for assembly. Gag targeting and assembly on the PM is dependent on interactions between its matrix (MA) domain and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂). Subsequent to Gag assembly, the envelope (Env) protein is recruited to the PM for incorporation into virus particles. Evidence suggests that the incorporation of the Env protein is mediated by interactions between the MA domain of Gag and the cytoplasmic tail of the gp41 subunit of Env (gp41CT), a mechanism that remains to be elucidated. Trimerization of the MA domain of Gag appears to be an obligatory step for this interaction. The interplay between gp41CT, the MA trimer, and the membrane has yet to be determined. Our lab has pioneered methods and approaches to investigate, at the molecular level, how the retroviral MA domains of Gag interact with membranes, a key requirement for understanding the Gag assembly and Env incorporation. Herein, we devised innovative approaches that will enable the structural characterization of the gp41CT–MA–membrane interactions. We employed structural biology (NMR and cryo-electron microscopy, biophysical methods, and biochemical tools to generate a macromolecular picture of how the MA domain of Gag binds to the membrane and how it interacts with gp41CT. To this end, we: (i) determined the three-dimensional structure of HIV-1 gp41CT and characterized its interaction with the membrane, (ii) engineered trimeric constructs of gp41CT and the MA to recapitulate the native and functional states of the proteins, and (iii) utilized membrane nanodisc technology to anchor the MA and gp41CT proteins. Our studies will allow for a detailed structural characterization of the gp41CT–MA–membrane interactions, which will advance our knowledge of HIV-1 Gag assembly and Env incorporation. Full article

The omics disciplines remain largely distinct sciences due to the necessity of separating molecular classes for different assays. For example, water-soluble and lipid bilayer-bound proteins and metabolites are usually studied separately. Nonetheless, it is at the interface between these sciences where biology happens. That is, lipid-interacting proteins typically recognize and transduce signals and regulate the flow of metabolites in the cell. Technologies are emerging to converge the omics. It is now possible to separate intact membrane:protein assemblies (memteins) directly from intact cells or cell membranes. Such complexes mediate complete metabolon, receptor, channel, and transporter functions. The use of poly(styrene-co-maleic acid) (SMA) copolymers has allowed their separation in a single step without any exposure to synthetic detergents or artificial lipids. This is a critical development as these agents typically strip away biological lipids, signals, and metabolites from their physiologically-relevant positions on proteins. The resulting SMA lipid particles (SMALPs) represent native nanodiscs that are suitable for elucidation of structures and interactions that occur in vivo. Compatible tools for resolving the contained memteins include X-ray diffraction (XRD), cryo-electron microscopy (cryoEM), mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy. Recent progress shows that memteins are more representative than naked membrane proteins devoid of natural lipid and is driving the development of next generation polymers. Full article