Nickel-chelating Lipid Research Articles

Calcium Signaling in T Cells Is Induced by Binding to Nickel-Chelating Lipids in Supported Lipid Bilayers.

Supported lipid bilayers (SLBs) are one of the most common cell-membrane model systems to study cell-cell interactions. Nickel-chelating lipids are frequently used to functionalize the SLB with polyhistidine-tagged ligands. We show here that these lipids by themselves can induce calcium signaling in T cells, also when having protein ligands on the SLB. This is important to avoid “false” signaling events in cell studies with SLBs, but also to better understand the molecular mechanisms involved in T-cell signaling. Jurkat T cells transfected with the non-signaling molecule rat CD48 were found to bind to ligand-free SLBs containing ≥2 wt% nickel-chelating lipids upon which calcium signaling was induced. This signaling fraction steadily increased from 24 to 60% when increasing the amount of nickel-chelating lipids from 2 to 10 wt%. Both the signaling fraction and signaling time did not change significantly compared to ligand-free SLBs when adding the CD48-ligand rat CD2 to the SLB. Blocking the SLB with bovine serum albumin reduced the signaling fraction to 11%, while preserving CD2 binding and the exclusion of the phosphatase CD45 from the cell-SLB contacts. Thus, CD45 exclusion alone was not sufficient to result in calcium signaling. In addition, more cells signaled on ligand-free SLBs with copper-chelating lipids instead of nickel-chelating lipids and the signaling was found to be predominantly via T-cell receptor (TCR) triggering. Hence, it is possible that the nickel-chelating lipids act as ligands to the cell’s TCRs, an interaction that needs to be blocked to avoid unwanted cell activation.

Kinetics of Histidine-Tagged Protein Association to Nickel-Decorated Liposome Surfaces.

Nickel-chelating lipids offer a convenient platform for reversible immobilization of histidine-tagged proteins to liposome surfaces. This interaction recently found utility as a model system for studying membrane remodeling triggered by protein crowding. Despite its wide array of utility, the molecular details of transient protein association to the lipid surfaces decorated with such chelator lipids remains poorly understood. In this study, we explore the kinetics of protein-liposome association across a wide concentration range using stopped-flow fluorescence. The fluorescence of histidine-tagged protein containing an intrinsic fluorophore (superfolder green fluorescent protein, SfGFP) was quenched upon binding to Ni-NTA-modified liposomes containing the quencher Dabsyl-PE lipids. Stopped-flow fluorescence reveals a complex, multiexponential binding behavior with a fast (kobs ∼ 10-20 s-1) phase and slower (kobs < 4 s-1) phase. Interestingly, the observed rates for the slower phase increase initially under low concentrations but start decreasing once a critical concentration is reached. Despite differences in the binding time scales, we observe that the trend of decreasing rates is reproducible irrespective of the chelator lipid doping level, protein surface charge, or lipid composition. Consideration of the protein footprint and membrane surface area occupancy leads us to conclude that the multiphasic binding behavior is reflective of protein binding via two distinct binding conformations. We propose that preliminary steps in protein association involve binding of a sterically occlusive side-on conformation followed by reorganization that leads to an end-on conformation with increased packing density. These results are important for the improvement of histidine-tag-based immobilization strategies and offer mechanistic insight into intermediates preceding membrane bending driven by protein crowding.

Development and characterization of stabilized, polymerized phospholipid bilayers on silica particles for specific immobilization of His-tagged proteins

Stabilized phospholipid bilayer (PLB) coated silica microspheres were prepared via polymerization of lipid monomers. These lipid coated silica microspheres are stable to both extended storage in solution at 4∘C and dry storage at room temperature. These stabilized lipid coated microspheres have also been functionalized with nickel-chelating lipids, a commonly used tool for immobilizing polyhistidine-tagged proteins. It is shown that 6xHis-EGFP interacts with (poly)bis-SorbPC/DOGS-NTA-Ni2+ coated silica and this interaction was interrupted by washing with imidazole indicating the reversibility of the interaction. No interaction was observed between the functionalized silica substrate and EGFP, which lacks the 6xHis-tag. Furthermore, these biocompatible (poly)bis-SorbPC coated microspheres were able to minimize non-specific protein adsorption. Biocompatible and highly stable phospholipid bilayers supported on silica microspheres were prepared by UV polymerization of a synthetic lipid. The resulting polymeric coatings were stable to extended storage in both solution and dry conditions. Furthermore, these biocompatible polymeric phospholipid bilayers allow incorporation of biologically and chemically important chemical moieties such as NTA-Ni2+ and exhibited enhanced capability of reducing nonspecific protein adsorption. X-axis in the figure is fluorescence intensity of GFP in a.u.

Label‐Free Measurements of the Diffusivity of Molecules in Lipid Membranes

An important and characteristic property of a cell membrane is the lateral mobility of protein molecules in the lipid bilayer. This has conventionally been measured by labeling the molecules with fluorescent markers and monitoring their mobility by different fluorescence-based techniques. However, adding the label to the studied molecule may affect the system, so it is an assumption in almost all experiments that the measured mobility of the biomolecule with its label is the same as that of the unlabeled molecule. However, this assumption is rarely tested due to a lack of suitable methods. In this work, a new technique to perform label-free diffusivity measurements is developed and used to measure the effect of the label for two common protein-lipid systems: 1) streptavidin (SA) coupled to a supported lipid bilayer (SLB) through biotinylated lipids and 2) the extracellular part of the T-cell adhesion protein CD2, coupled to an SLB through histidine tags to nickel-chelating lipids. A measurable (≈12%) decrease in diffusivity is found for both labeled proteins, even though the molecular mass of the label is almost 100 times smaller than those of the proteins (≈50 kDa). The results illustrate the importance of being able to study different biophysical properties of cell membranes and their mimics without relying on fluorescent labels, especially if fluorescent labeling is difficult or is expected to affect the nature of the intermolecular interactions being studied.

Membrane Association of a Protein Increases the Rate, Extent, and Specificity of Chemical Cross-Linking

Many cellular processes involve interactions between membrane-associated proteins, and those interactions are enhanced by membrane association. We have used cross-linking reactions to compare the extent and specificity of protein interactions in solution versus on a membrane surface. Cysteine mutants of a soluble cytoplasmic fragment (CF) of the aspartate receptor, a transmembrane receptor involved in bacterial chemotaxis, are used in disulfide bond formation with the thiol-specific oxidant diamide and chemical cross-linking reactions with the trifunctional maleimide TMEA. CF binding to membranes is mediated by its N-terminal His tag binding to vesicles containing a nickel-chelating lipid, so cross-linking reactions conducted in the presence and absence of vesicles differ only in whether CF is bound to the vesicles or is free in solution. For multiple Cys throughout the CF, membrane association is shown to increase the rate and extent of these reactions. Cross-linking specificity, which is measured as the preference for cross-linking between Cys near each other in the native structure, is also enhanced by membrane association. These results provide an experimental demonstration that membrane binding enhances protein-protein interactions, an important consideration for understanding processes involving membrane-associated proteins. The experiments further demonstrate the importance of cross-linking conditions for these reactions that are often used to probe protein structure and dynamics and the potential of membrane association to restore native interactions of membrane-associated proteins for cross-linking studies.

Modeling immune receptor clustering with nano-scale discoidal membrane bilayers (P3350)

Abstract MHC proteins on antigen-presenting cells form clusters with each other and other cell surface ligands. The changes in the clustering serve as a sensitive mechanism to modulate T cell responses. We propose to mimic MHC membrane patches using nanolipoprotein particles (NLPs). NLPs are nano-scale discoidal lipid bilayers stabilized by apolipoproteins. Nickel-chelating lipids incorporated into the NLP bilayer capture soluble polyhistidine-tagged pMHC. We have studied the interactions of cognate (non-self) and non-cognate (self) pMHC/NLPs with CD8+ CTL. Both self and non-self pMHC/NLPs were bound to the T-cell surface but to different extents. The apparent equilibrium binding constants of both non-self and self pMHC/NLPs were strongly dependent on pMHC density. A 3-fold difference in pMHC density (from 10:1 to 30:1 per NLP) caused 10-fold and 30-fold increases in the apparent affinity, respectively. Surprisingly, the avidity of self and non-self multivalent interactions appears to be similar when the pMHC density on the NLP surface is high. The similar avidity may be explained by a very rapid on-rate of CD8 binding to clustered MHC and marginal difference in the dissociation rates at the high epitope density resulting from very efficient CD8-MHC rebinding. NLP-based model MHC clusters with pre-determined properties could be incorporated onto glass beads coated with a lipid bilayer to engineer artificial APC with the goal of inducing ex vivo long-lived T-cell effectors for adoptive T-cell therapy.

Adjuvant and antigen co-delivery with nickel-chelating lipid nanoparticles

Adjuvant and antigen co-delivery with nickel-chelating lipid nanoparticles

A novel method of AquaporinZ incorporation via binary-lipid Langmuir monolayers

In this work, a new approach of incorporating transmembrane protein AquaporinZ (AqpZ) into a lipid bilayer has been developed with the aid of the Langmuir–Blodgett technique. The binary-lipid monolayer for AqpZ incorporation is composed of (1) gel-phase lipids resistant to detergent dissolution and (2) nickel-chelating lipids that can attach the histidine-tagged AqpZ from the subphase. Upon removal of subphase detergent with BioBeads, the incorporation is achieved by transferring the AqpZ-associated binary-lipid monolayer onto a preformed pure binary-lipid monolayer using the Langmuir–Schaefer deposition method. AFM images show an indication of AqpZ incorporation in the bilayer. Furthermore, it is also shown that BioBeads can remove a significant amount of detergent in the subphase and lipid film integrity is restored after detergent removal. The detergent removal rate is correlated to BioBeads amount and subphase circulation. The new approach of AqpZ reconstitution revealed in this work could potentially be applied in biomimetic membrane formation for water purification applications.

Nanolipoprotein particles as vaccine platforms for co-localization of adjuvants and subunit antigens (106.10)

Abstract Subunit vaccines provide a number of important benefits over traditional vaccine candidates, such as overall safety. However, these antigens require immunostimulatory adjuvants to generate the appropriate adaptive immune response. We hypothesize that the efficacy of subunit antigens will be enhanced if co-localized with adjuvant during delivery in a single delivery platform. Thus, our goal is to develop a robust vaccine delivery platform that will allow for the co-delivery of both subunit antigens and adjuvants using nanolipoprotein particles (NLPs). NLPs are discoidal HDL analogs comprised of lipid bilayers stabilized by scaffold proteins. These biocompatible, nanoscale particles can be assembled to incorporate nickel-chelating lipids to form nickel-chelating NLPs (NiNLPs). Thus, any His-tagged antigens can be conjugated to the NiNLPs. The amphipathic adjuvants monophosphoryl Lipid A (MPLA) and cholesterol-modified CpGs (Chol-CpGs) were anchored to the lipid bilayer of the NiNLPs. His-tagged influenza hemagglutinin 5 was conjugated to adjuvant:NiNLPs and injected into mice. Antibody titers increased 32 and 8 fold (MPLA and CpG, respectively) compared to co-administered formulations. Also, the immune response was skewed towards a Th1 response, with up to 16-fold increases in IgG2a:IgG1 ratios. Our findings demonstrate that co-localized delivery of adjuvant with antigens provides significantly greater immune stimulation in mice compared to co-administered formulations.

Study of the PDK1/AKT signaling pathway using selective PDK1 inhibitors, HCS, and enhanced biochemical assays

The PI3K/AKT signaling pathway has an important regulatory role in cancer cell growth and tumorigenesis. Signal transduction through this pathway requires the assembly and activation of PDK1 and AKT at the plasma membrane. On activation of the pathway, PDK1 and AKT1/2 translocate to the membrane and bind to phosphatidylinositol-(3,4,5)-trisphosphate (PIP 3) through interaction with their pleckstrin-homology domains. A biochemical method was developed to measure the kinase activity of PDK1 and AKT1/2, utilizing nickel-chelating coated lipid vesicles as a way to mimic the membrane environment. The presence of these vesicles in the reaction buffer enhanced the specific activity of the His-tagged PDK1 (full-length, and the truncated kinase domain) and the activity of the full-length His-tagged AKT1 and AKT2 when assayed in a cascade-type reaction. This enhanced biochemical assay is also suitable for measuring the inhibition of PDK1 by several selective compounds from the carbonyl-4-amino-pyrrolopyrimidine (CAP) series. One of these inhibitors, PF-5168899, was further evaluated using a high content cell-based assay in the presence of CHO cells engineered with GFP-PDK1.

Metallochelating liposomes with associated lipophilised norAbuMDP as biocompatible platform for construction of vaccines with recombinant His-tagged antigens: Preparation, structural study and immune response towards rHsp90

Hsp90-CA is present in cell wall of Candida pseudohyphae or hyphae — typical pathogenic morphotype for both systemic and mucosal Candida infections. Heat shock protein from Candida albicans (hsp90-CA) is an important target for protective antibodies during disseminated candidiasis of experimental mice and human. His-tagged protein rHsp90 was prepared and used as the antigen for preparation of experimental recombinant liposomal vaccine. Nickel-chelating liposomes (the size around 100 nm, PDI ≤ 0.1) were prepared from the mixture of egg phosphatidyl choline and nickel-chelating lipid DOGS-NTA-Ni (molar ratio 95:5%) by hydration of lipid film and extrusion methods. New non-pyrogenic hydrophobised derivative of MDP (C18-O-6-norAbuMDP) was incorporated into liposomes as adjuvans. rHsp90 was attached onto the surface of metallochelating liposomes by metallochelating bond and the structure of these proteoliposomes was studied by dynamic light scattering, AF microscopy, TEM and GPC. The liposomes with surface-exposed C18-O-6-norAbuMDP were well recognised and phagocyted by human dendritic cells in vitro. In vivo the immune response towards this experimental vaccine applied in mice (i.d.) demonstrated both TH1 and TH2 response comparable to FCA, but without any side effects. Metallochelating liposomes with lipophilic derivatives of muramyl dipeptide represent a new biocompatible platform for construction of experimental recombinant vaccines and drug-targeting systems.

Immobilization of histidine-tagged proteins on monodisperse metallochelation liposomes: Preparation and study of their structure

Liposomes represent a biocompatible platform for the construction of self-assembling proteoliposomes using nickel or zinc metallochelation. Potential applications of such structures consist in the development of new biocompatible vaccination nanoparticles and drug delivery nanoparticle systems. Here, we describe the design and construction of a flow-through ultrafiltration cell suitable for the preparation of monodisperse liposomes enabled for metallochelation and, hence, the formation of proteoliposomes. The linkage of the cell with a fast protein liquid chromatography system facilitates automation of the procedure, which fits the criteria for upscaling. Proof-of-concept experiments are performed using a mixture of egg phosphatidyl choline and nickel-chelating lipid DOGS–NTA–Ni (1,2-dioleoyl- sn-glycero-3-{[ N(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl}(nickel salt)) to formulate proteoliposomes with proteins attached by metallochelation, including histidine (His)-tagged recombinant green fluorescent protein and rgp120 (derived from HIV-1 Env). These model proteoliposomes are characterized by gel permeation chromatography and by dynamic light scattering. Transmission electron microscopy and immunogold staining are used to characterize surface-bound proteins, revealing the tendency of rgp120 to form microdomains on liposome surfaces. These microdomains possess a two-dimensional crystal-like structure that is seen more precisely by atomic force microscopy.

Pattern Formation and Molecular Transport of Histidine-Tagged GFPs Using Supported Lipid Bilayers

We fabricated a heterogeneous supported lipid bilayer (SLB) by employing binary lipid mixtures comprising a saturated acyl chain DSPC and an unsaturated acyl chain nickel-chelating lipid. By using the specific adsorption properties of histidine-tagged proteins (His-tagged GFPs) in relation to nickel-chelating lipids, we demonstrated protein pattern formation on the SLB corresponding to the phase separation pattern of the SLB. In addition, by using a lipid mixture consisting of an unsaturated acyl chain DOPC and a nickel-chelating lipid, and His-tagged GFPs, we succeeded in transporting the proteins along a hydrophilic micropattern on a SiO(2) substrate. The protein transport is induced by the self-spreading behavior of a fluid SLB with a kinetic spreading coefficient beta = 10.4 microm(2) s(-1). This method provides a guide for strategically carrying various biomolecules to specific positions by using a soft biointerface on a solid surface. In addition, the results demonstrate the importance of using techniques that allow the controlled manipulation of biomolecules based on the static or dynamic properties of the SLB platform.

Kinetic Analysis of His-Tagged Protein Binding to Nickel-Chelating Nanolipoprotein Particles

Nanolipoprotein particles (NLPs) are discoidal self-assembling membrane mimetics that have been primarily used as a platform for the solubilization and stabilization of membrane proteins. Nickel-chelating nanolipoprotein particles (NiNLPs) containing nickel-chelating lipids (Ni-lipid) for the targeted immobilization of His-tagged proteins hold promise as carriers of hydrophilic biological molecules for a range of applications. The effect of protein loading (i.e., the number of proteins bound per NiNLP) and Ni-lipid content on the time scales and kinetics of binding are important to various applications such as vaccine development, diagnostic imaging, and drug delivery. We have immobilized hexa-His-tagged LsrB, a Yersinia pestis transport protein, onto NiNLPs to examine the effect of protein binding stoichiometry and Ni-lipid content on the time scales and kinetics of protein binding by surface plasmon resonance (SPR). Data indicate that the dissociation half-time increases with Ni-lipid content up to a molar concentration of 35% and decreases as the number of bound protein per NiNLP increases. These findings indicate that the kinetics of protein binding are highly dependent on both the number of bound protein per NiNLP and Ni-lipid content.

Abstract 3122: Her4 and Her2/neu tyrosine kinase domains dimerize and activate in a reconstituted in vitro system

Abstract Her4 (ErbB-4) and Her2/neu (ErbB-2) are receptor tyrosine kinases belonging to the Epidermal Growth Factor Receptor (EGFR) family. Crystal structures of EGFR and Her4 kinase domains have demonstrated kinase dimerization and activation through an allosteric mechanism. The kinase domains form an asymmetric dimer, where the C-lobe surface of one monomer contacts the N-lobe of the other monomer. In vitro studies of EGFR tyrosine kinase dimerization and activation were previously reported using a nickel-chelating lipid - liposome system, and we now demonstrate this system can be broadly applied to all other members of the EGFR family. Polyhistidine tagged-Her4, Her2/neu, and Her3 kinase domains are bound to these nickel-chelating lipid containing liposomes and are brought to a high local concentration, mimicking what happens to the full-length receptors in vivo following ligand binding. Addition of Nickel-liposomes to Her4 kinase domain results in a 40-fold activation in kinase specific activity and a marked enhancement in C-terminal tail autophosphorylation. Activation of Her4 shows a sigmoidal dependence on kinase concentration, consistent with a cooperative process that requires kinase dimerization. Her2/neu kinase activity is also activated by Nickel-liposomes, and further increases in Her2/neu activity are achieved by heterodimerization with either Her3 or Her4. The ability of Her3 and Her4 to heterodimerize and activate other family members is studied in vitro. Her3 kinase domain readily activates Her2/neu but is a poor activator of Her4, which differs from the prediction made by the asymmetric dimer model, and suggests that the mode of dimerization of Her3 needs to be further studied. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3122.

AFM study of the interaction of cytochrome P450 2C9 with phospholipid bilayers

Cytochromes P450 (CYP) are key enzymes involved in the metabolism of drugs and other lipophilic xenobiotics and endogenous compounds. In this study, atomic force microscopy was applied to characterise the association of CYP2C9 to dimyristoylphosphatidylcholine (DMPC) supported phospholipid bilayers. CYP2C9 was found to exclusively localise in the gel domains of partially melted DMPC bilayers. Despite lacking the N-terminus transmembrane spanning domain, the CYP2C9 protein appeared to partially embed into the membrane bilayer, as evidenced by an increase in melting temperature of surrounding phospholipids. Reversible binding of CYP2C9 via an engineered His tag to a phospholipid bilayer was facilitated using nickel-chelating lipids, presenting potential applications for biosensor technologies.

Interactions of the GM2 Activator Protein with Phosphatidylcholine Bilayers: A Site-Directed Spin-Labeling Power Saturation Study

The GM2 activator protein (GM2AP) is an accessory protein that is an essential component in the catabolism of the ganglioside GM2. A function of GM2AP is to bind and extract GM2 from intralysosomal vesicles, forming a soluble protein-lipid complex, which interacts with the hydrolase Hexosaminidase A, the enzyme that cleaves the terminal sugar group of GM2. Here, we used site-directed spin labeling with power saturation electron paramagnetic resonance to determine the surface-bound orientation of GM2AP upon phosphatidylcholine vesicles. Because GM2AP extracts lipid ligands from the vesicle and is undergoing exchange on and off the vesicle surface, we utilized a nickel-chelating lipid to localize the paramagnetic metal collider to the lipid bilayer-aqueous interface. Spin-labeled sites that collide with the lipid-bound metal relaxing agent provide a means for mapping sites of the protein that interact with the lipid bilayer interface. Results show that GM2AP binds to lipid bilayers such that the residues lining the lipid-binding cavity lie on the vesicle surface. This orientation creates a favorable microenvironment that can allow for the lipid tails to flip out of the bilayer directly into the hydrophobic pocket of GM2AP.

Immobilization of His-Tagged Proteins on Nickel-Chelating Nanolipoprotein Particles

Nanolipoprotein particles (NLPs) are nanometer-sized, discoidal particles that self-assemble from purified apolipoprotein and phospholipid. Their size and facile functionalization suggest potential application of NLPs as platforms for the presentation and delivery of recombinant proteins. To this end, we investigated incorporation of nickel-chelating lipids into NLPs (NiNLPs) and subsequent sequestration of polyhistidine (His)-tagged proteins. From initial lipid screens for NLP formation, the two phospholipids DMPC and DOPC were identified as suitable bulk lipids for incorporation of the nickel-chelating lipid DOGS-NTA-Ni into NLPs, and NiNLPs were successfully formed with varying amounts of DOGS-NTA-Ni. NiNLPs consisting of 10% DOGS-NTA-Ni with 90% bulk lipid (either DMPC or DOPC) were thoroughly characterized by size exclusion chromatography (SEC), non-denaturing gradient gel electrophoresis (NDGGE), and atomic force microscopy (AFM). Three different His-tagged proteins were sequestered on NiNLPs in a nickel-dependent manner, and the amount of immobilized protein was contingent on the size and composition of the NiNLP.

Synthesis of Nickel-Chelating Fluorinated Lipids for Protein Monolayer Crystallizations

Nickel-chelating lipids have been synthesized for use as functionalized templates for 2-D crystallization of membrane proteins. These monolayer-forming lipids have been designed with three distinct components: (i) a branched hydrocarbon tail to confer fluidity of the monolayer, (ii) a perfluorinated central core for detergent resistance, and (iii) a nickel-chelating hydrophilic headgroup to facilitate binding of recombinant, polyhistidine-tagged fusion proteins. Alkylations of fluorinated alcohols used in these syntheses proceed in good yields only with the application of prolonged sonication and, in some cases, in the presence of phase-transfer catalysts. Formation of 2-D crystals of the His-tagged membrane protein BmrA from Bacillus subtilis is reported.

Kinetic Control of Histidine-Tagged Protein Surface Density on Supported Lipid Bilayers

Nickel-chelating lipids are general tools for anchoring polyhistidine-tagged proteins to supported lipid bilayers (SLBs), but controversy exists over the stability of the protein-lipid attachment. Here, we show that chelator lipids are suitable anchors for building stable, biologically active surfaces but that a simple Langmuirian model is insufficient to describe their behavior. Desorption kinetics from chelator lipids are governed by the valency of surface binding: monovalently bound proteins desorb within minutes (t1/2 approximately 6 min), whereas polyvalently bound species remain bound for hours (t1/2 approximately 12 h). Evolution between surface states is slow, so equilibrium is unlikely to be reached on experimental timescales. However, by tuning incubation conditions, the populations of each species can be kinetically controlled, providing a wide range of protein densities on SLBs with a single concentration of chelator lipid. We propose guidelines for the assembly of SLB surfaces functionalized with specific protein densities and demonstrate their utility in the formation of hybrid immunological synapses.