Identifying Membrane Protein-Lipid Interactions with Lipidomic Lipid Exchange-Mass Spectrometry.

Lipids can play important roles in modulating membrane protein structure and function. However, it is challenging to identify natural lipids bound to membrane proteins in complex bilayers. Here, we developed lipidomic lipid exchange-mass spectrometry (LX-MS) to study the lipid affinity for membrane proteins on a lipidomic scale. We first mix membrane protein nanodiscs with empty nanodiscs that have no embedded membrane proteins. After allowing lipids to passively exchange between the two populations, we separate the two types of nanodiscs and perform lipidomic analysis on each with liquid chromatography and MS. Enrichment of lipids in the membrane protein nanodiscs reveals the affinity of individual lipids for binding the target membrane protein. We apply this approach to study three membrane proteins. With the Escherichia coli ammonium transporter AmtB and aquaporin AqpZ in nanodiscs with E. coli polar lipid extracts, we detected binding of cardiolipin and phosphatidyl-glycerol lipids to the proteins. With the acetylcholine receptor in nanodiscs with brain polar lipid extracts, we discovered a complex set of lipid interactions that depended on the head group and tail composition. Overall, lipidomic LX-MS provides a detailed understanding of the lipid-binding affinity and thermodynamics for membrane proteins in complex bilayers and provides a unique perspective on the chemical environment surrounding membrane proteins.

Hybrid Nanoparticle Platform for Nanoscale Scintillation Proximity Assay.

ß-particle emitting radionuclides, such as 3H, 14C, 32P, 33P, and 35S, are important molecular labels due to their small size and the prevalence of these atoms in biomolecules but are challenging to selectively detect and quantify within aqueous biological samples and systems. Here, we present a core-shell nanoparticle-based scintillation proximity assay platform (nanoSPA) for the separation-free, selective detection of radiolabeled analytes. nanoSPA is prepared by incorporating scintillant fluorophores into polystyrene core particles and encapsulating the scintillant-doped cores within functionalized silica shells. The functionalized surface enables covalent attachment of specific binding moieties such as small molecules, proteins, or DNA that can be used for analyte-specific detection. nanoSPA was demonstrated for detection of 3H-labeled analytes, the most difficult biologically relevant ß-emitter to measure due to the low energy ß-particle emission, using three model assays that represent covalent and non-covalent binding systems that necessitate selectivity over competing 3H-labeled species. In each model, nmol quantities of target were detected directly in aqueous solution without separation from unbound 3H-labeled analyte. The nanoSPA platform facilitated measurement of 3H-labeled analytes directly in bulk aqueous samples without surfactants or other agents used to aid particle dispersal. Selectivity for bound 3H-analytes over unbound 3H analytes was enhanced up to 30-fold when the labeled species was covalently bound to nanoSPA, and 4- and 8-fold for two non-covalent binding assays using nanoSPA. The small size and enhanced selectivity of nanoSPA should enable new applications compared to the commonly used microSPA platform, including the potential for separation-free, analyte-specific cellular or intracellular detection.

Enhanced Fluorescent Protein Activity in Polymer Scaffold-Stabilized Phospholipid Nanoshells Using Neutral Redox Initiator Polymerization Conditions.

Phospholipid nanoshells, for example, liposomes, provide a versatile enabling platform for the development of nanometer-sized biosensors and molecular delivery systems. Utilization of phospholipid nanoshells is limited by the inherent instability in complex biological environments, where the phospholipid nanoshell may disassemble and degrade, thus releasing the contents and destroying sensor function. Polymer scaffold stabilization (PSS), wherein the phospholipid nanoshells are prepared by partitioning reactive monomers into the lipid bilayer lamella followed by radical polymerization, has emerged to increase phospholipid nanoshell stability. In this work, we investigated the effects of three different radical initiator conditions to fabricate stable PSS-phospholipid nanoshells yet retain the activity of encapsulated model fluorescent sensor proteins. To identify nondestructive initiation conditions, UV photoinitiation, neutral redox initiation, and thermal initiation were investigated as a function of PSS-phospholipid nanoshell stabilization and fluorescence emission intensity of enhanced green fluorescent protein (eGFP) and tandem dimer Tomato (td-Tomato). All three initiator approaches yielded comparably stable PSS-phospholipid nanoshells, although slight variations in PSS-phospholipid nanoshell size were observed, ranging from ca. 140 nm for unstabilized phospholipid nanoshells to 300-500 nm for PSS-phospholipid nanoshells. Fluorescence emission intensity of encapsulated eGFP was completely attenuated under thermal initiation (0% vs control), moderately attenuated under UV photoinitiation (40 ± 4% vs control), and unaffected by neutral redox initiation (97 ± 3% vs control). Fluorescence emission intensity of encapsulated td-Tomato was significantly attenuated under thermal initiation (13 ± 3% vs control), moderately attenuated UV photoinitiation (64 ± 5% vs control), and unaffected by neutral redox initiation (98% ± 4% vs control). Therefore, the neutral redox initiation method provides a significant advancement toward the preparation of protein-functionalized PSS-phospholipid nanoshells. These results should help to guide future applications and designs of biosensor platforms using PSS-phospholipid nanoshells and other polymer systems employing protein transducers.

Expression, purification, and electrophysiological characterization of a recombinant, fluorescent Kir6.2 in mammalian cells.

The inwardly rectifying K+ (Kir) channel, Kir6.2, plays critical roles in physiological processes in the brain, heart, and pancreas. Although Kir6.2 has been extensively studied in numerous expression systems, a comprehensive description of an expression and purification protocol has not been reported. We expressed and characterized a recombinant Kir6.2, with an N-terminal decahistidine tag, enhanced green fluorescent protein (eGFP) and deletion of C-terminal 26 amino acids, in succession, denoted eGFP-Kir6.2Δ26. eGFP-Kir6.2Δ26 was expressed in HEK293 cells and a purification protocol developed. Electrophysiological characterization showed that eGFP-Kir6.2Δ26 retains native single channel conductance (64 ±â€¯3.3 pS), mean open times (τ1 = 0.72 ms, τ2 = 15.3 ms) and ATP affinity (IC50 = 115 ±â€¯25 µM) when expressed in HEK293 cells. Detergent screening using size exclusion chromatography (SEC) identified Fos-choline-14 (FC-14) as the most suitable surfactant for protein solubilization, as evidenced by maintenance of the native tetrameric structure in SDS-PAGE and western blot analysis. A two-step scheme using Co2+-metal affinity chromatography and SEC was implemented for purification. Purified protein activity was assessed by reconstituting eGFP-Kir6.2Δ26 in black lipid membranes (BLMs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), l-α-phosphatidylinositol-4,5-bisphosphate (PIP2) in a 89.5:10:0.5 mol ratio. Reconstituted eGFP-Kir6.2Δ26 displayed similar single channel conductance (61.8 ±â€¯0.54 pS) compared to eGFP-Kir6.2Δ26 expressed in HEK293 membranes; however, channel mean open times increased (τ1 = 7.9 ms, τ2 = 61.9 ms) and ATP inhibition was significantly reduced for eGFP-Kir6.2Δ26 reconstituted into BLMs (IC50 = 3.14 ±â€¯0.4 mM). Overall, this protocol should be foundational for the production of purified Kir6.2 for future structural and biochemical studies.

Polystyrene-Core, Silica-Shell Scintillant Nanoparticles for Low-Energy Radionuclide Quantification in Aqueous Media.

ß-particle emitting radionuclides are useful molecular labels due to their abundance in biomolecules. Detection of ß-emission from 3H, 35S, and 33P, important biological isotopes, is challenging due to the low energies (Emax ≤ 300 keV) and short penetration depths (≤0.6 mm) in aqueous media. The activity of biologically relevant ß-emitters is usually measured in liquid scintillation cocktail (LSC), a mixture of energy-absorbing organic solvents, surfactants, and scintillant fluorophores, which places significant limitations on the ability to acquire time-resolved measurements directly in aqueous biological systems. As an alternative to LSC, we developed polystyrene-core, silica-shell nanoparticle scintillators (referred to as nanoSCINT) for quantification of low-energy ß-particle emitting radionuclides directly in aqueous solutions. The polystyrene acts as an absorber for energy from emitted ß-particles and can be loaded with a range of hydrophobic scintillant fluorophores, leading to photon emission at visible wavelengths. The silica shell serves as a hydrophilic shield for the polystyrene core, enabling dispersion in aqueous media and providing better compatibility with water-soluble analytes. While polymer and inorganic scintillating microparticles are commercially available, their large size and/or high density complicates effective dispersion throughout the sample volume. In this work, nanoSCINT nanoparticles were prepared and characterized. nanoSCINT responds to 3H, 35S, and 33P directly in aqueous solutions, does not exhibit a change in scintillation response between pH 3.0 and 9.5 or with 100 mM NaCl, and can be recovered and reused for activity measurements in bulk aqueous samples, demonstrating the potential for reduced production of LSC waste and reduced total waste volume during radionuclide quantification. The limits of detection for 1 mg/mL nanoSCINT are 130 nCi/mL for 3H, 8 nCi/mL for 35S, and <1 nCi/mL for 33P.

Composite nanoparticles: the best of two worlds.

Nanomaterials have rapidly moved into the mainstream for chemical and biological analysis. Nanoparticle probes enhance signal intensity, increase the chemical and physical stability of the probe, and facilitate surface modification for specific targeting. Unfortunately, common problems are encountered with many nanoparticle probes, e.g., poor solubility, poor biocompatibility, and leakage of encapsulated components, that severely restrict the application of probes to ex vivo samples under carefully controlled conditions. A wide range of recently developed multifunctional nanomaterials are poised to make significant contributions to molecular analysis of biological systems. Composite nanoparticle geometries, including composites, hybrids, and core-shell nanoparticles prepared using two or more materials, e.g., silica/inorganic, silica/polymer, or polymer/inorganic combinations, offer improved solubility, easier functionalization, and decreased toxicity compared with the related single-component materials. Furthermore, composite nanomaterials present substantial signal amplification, and improved multiplexing for higher-sensitivity and higher-resolution measurements. Further development and integration of composite nanomaterials into the quantitative sciences will play a key role in the future of functional probes for imaging, quantitative analysis, and biological manipulation.

Polymer-stabilized phospholipid vesicles with a controllable, pH-dependent disassembly mechanism.

In this letter, we report a facile method to prepare robust phospholipid vesicles using commonly available phospholipids that are stabilized via the formation of an interpenetrating, acid-labile, cross-linked polymer network that imparts a site for controlled polymer destabilization and subsequent vesicle degradation. The polymer network was formed in the inner lamella of the phospholipid bilayer using 2,2-di(methacryloyloxy-1-ethoxy)propane (DMOEP) and butyl methacrylate (BMA). Upon exposure to acidic conditions, the highly cross-linked polymer network was partially converted to smaller linear polymers, resulting in substantially reduced vesicle stability upon exposure to chemical and physical insults. Isolated polymers had pH-dependent-solubility in THF. Transmission electron microscopy and dynamic light scattering revealed time-dependent enhanced vesicle stability in high concentrations of surfactant and vacuum conditions at elevated pH, whereas exposure to acidic pH rapidly decreased the vesicle stability, with complete destabilization observed in less than 24 h. The resultant transiently stabilized vesicles may prove useful for enhanced drug delivery and chemical sensing applications and allow for improved physiological clearance.