fabrication-procedures-birefringence-measurements-for-designing

Fabrication Procedures and Birefringence Measurements for Designing Magnetically Responsive Lanthanide Ion Chelating Phospholipid Assemblies

Bicelles are tunable disk-like polymolecular assemblies formed from a large variety of lipid mixtures. Applications range from membrane protein structural ...

ETH Zurich

We find that the putative Diels-Alderase macrophomate synthase (MPS) catalyzes the addition of pyruvate enolate, generated by decarboxylation of oxaloacetate, to a variety of aldehydes. Alkyl, aryl, and heteroaryl aldehydes are accepted as substrates, providing gamma-hydroxy-alpha-ketoacids in 35-95% yield with modest levels of stereochemical control. These aldol products, which are difficult to synthesize by other methods, are formed with efficiency comparable to that of macrophomate. Our results thus provide evidence that a two-step Michael-aldol pathway is a plausible alternative to the postulated [4 + 2] cycloaddition in the MPS-catalyzed addition of pyruvate enolate to 2-pyrones. They are also relevant to understanding the divergent evolution of type II pyruvate aldolases.

The putative Diels-Alderase macrophomate synthase is an efficient aldolase.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a fast analysis tool employed for the detection of a broad range of analytes. However, MALDI-MS has a reputation of not being suitable for quantitative analysis. Inhomogeneous analyte/matrix co-crystallization, spot-to-spot inhomogeneity, as well as a typically low number of replicates are the main contributing factors. Here, we present a novel MALDI sample target for quantitative MALDI-MS applications, which addresses the limitations mentioned above. The platform is based on the recently developed microarray for mass spectrometry (MAMS) technology and contains parallel lanes of hydrophilic reservoirs. Samples are not pipetted manually but deposited by dragging one or several sample droplets with a metal sliding device along these lanes. Sample is rapidly and automatically aliquoted into the sample spots due to the interplay of hydrophilic/hydrophobic interactions. With a few microliters of sample, it is possible to aliquot up to 40 replicates within seconds, each aliquot containing just 10 nL. The analyte droplet dries immediately and homogeneously, and consumption of the whole spot during MALDI-MS analysis is typically accomplished within few seconds. We evaluated these sample targets with respect to their suitability for use with different samples and matrices. Furthermore, we tested their application for generating calibration curves of standard peptides with Î±-cyano-4-hdydroxycinnamic acid as a matrix. For angiotensin II and [Glu(1)]-fibrinopeptide B we achieved coefficients of determination (r(2)) greater than 0.99 without the use of internal standards.

Self-Aliquoting Microarray Plates for Accurate Quantitative Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

Lanthanides have been used for several decades to increase the magnetic alignability of bicelles. DMPE-DTPA (1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine-diethylenetriaminepentaacetate) is commonly applied to anchor the lanthanides into the bicelles. However, because DMPE-DTPA has the tendency to accumulate at the highly curved edge region of the bicelles and if located there does not contribute to the magnetic orientation energy, we have tested cholesterol-DTPA complexed with thulium ions (Tm(3+)) as an alternative chelator to increase the magnetic alignability. Differential scanning calorimetric (DSC) measurements indicate the successful integration of cholesterol-DTPA into a DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) bilayer. Cryo transmission electron microscopy and small-angle neutron scattering (SANS) measurements show that the disklike structure, that is, bicelles, is maintained if cholesterol-DTPA·Tm(3+) is integrated into a mixture of DMPC, cholesterol, and DMPE-DTPA·Tm(3+). The size of the bicelles is increased compared to the size of the bicelles obtained from mixtures without cholesterol-DTPA·Tm(3+). Magnetic-field-induced birefringence and SANS measurements in a magnetic field show that with addition of cholesterol-DTPA·Tm(3+) the magnetic alignability of these bicelles is significantly increased compared to bicelles composed of DMPC, cholesterol, and DMPE-DTPA·Tm(3+) only.

Cholesterol-diethylenetriaminepentaacetate complexed with thulium ions integrated into bicelles to increase their magnetic alignability.

Mesostructures responding to external triggers such as temperature, pH, or magnetic field have the potential to be used as self-acting sensors, detectors, or switches. Key features are a strong and well-defined response to the external trigger. Here, we present magnetic alignable bicelles embedded into a gelatin matrix generating magnetically switchable structures, which can reversibly be locked and unlocked by adjusting the temperature. We show that the disk-like aggregates can be orientated in magnetic fields, and such orientation can be preserved after embedding into gelatin. The resulting gel cubes show an anisotropic transfer for electromagnetic waves, i.e., a different spatial birefringence. Cycling through the melting point of gelatin sets the structure back to its isotropic state providing a read-out of the thermal history. Stacking of the bicelles induced by the gelatin promotes magnetic aligning, as an increased aggregation number in the stacks increases the magnetic orientation energy.

Magnetically enhanced bicelles delivering switchable anisotropy in optical gels.

We present a robust droplet-based device, which enables the fractionation of ultralow flow rate nanoflow liquid chromatography (nano-LC) eluate streams at high frequencies and high peak resolution. This is achieved by directly interfacing the separation column to a micro T-junction, where the eluate stream is compartmentalized into picoliter droplets. This immediate compartmentalization prevents peak dispersion during eluate transport and conserves the chromatographic performance. Subsequently, nanoliter eluate fractions are collected at a rate of one fraction per second on a high-density microarray to retain the separation with high temporal resolution. Chromatographic separations of up to 45 min runtime can thus be archived on a single microarray possessing 2700 sample spots. The performance of this device is demonstrated by fractionating the separation of a tryptic digest of a known protein mixture onto the microarray chip and subsequently analyzing the sample archive using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Resulting peak widths are found to be significantly reduced compared to standard continuous flow spotting technologies as well as in comparison to a conventional nano-LC-electrospray ionization-mass spectrometry interface. Moreover, we demonstrate the advantage of our high-definition nanofractionation device by applying two different MALDI matrices to all collected fractions in an alternating fashion. Since the information that is obtained from a MALDI-MS measurement depends on the choice of MALDI matrix, we can extract complementary information from neighboring spots containing almost identical composition but different matrices.

High-resolution droplet-based fractionation of nano-LC separations onto microarrays for MALDI-MS analysis.

We present a novel and straightforward screening method to detect protein phosphorylations in complex protein mixtures. A proteolytic digest is separated by a conventional nanoscale liquid chromatography (nano-LC) separation and the eluate is immediately compartmentalized into microdroplets, which are spotted on a microarray MALDI plate. Subsequently, the enzyme alkaline phosphatase is applied to every second microarray spot to remove the phosphate groups from phosphorylated peptides, which results in a mass shift of n×-80 Da. The MALDI-MS scan of the microarray is then evaluated by a software algorithm to automatically identify the phosphorylated peptides by exploiting the characteristic chromatographic peak profile induced by the phosphatase treatment. This screening method does not require extensive MS/MS experiments or peak list evaluation and can be easily extended to other enzymatic or chemical reactions.

Screening for protein phosphorylation using nanoscale reactions on microdroplet arrays.

We demonstrate a new approach for the site-specific identification and characterization of protein N-glycosylation. It is based on a nano-liquid chromatography microarray-matrix assisted laser desorption/ionization-MS platform, which employs droplet microfluidics for on-plate nanoliter reactions. A chromatographic separation of a proteolytic digest is deposited at a high frequency on the microarray. In this way, a short separation run is archived into thousands of nanoliter reaction cavities, and chromatographic peaks are spread over multiple array spots. After fractionation, each other spot is treated with PNGaseF to generate two correlated traces within one run, one with treated spots where glycans are enzymatically released from the peptides, and one containing the intact glycopeptides. Mining for distinct glycosites is performed by searching for the predicted deglycosylated peptides in the treated trace. An identified peptide then leads directly to the position of the "intact" glycopeptide clusters, which are located in the adjacent spots. Furthermore, the deglycosylated peptide can be sequenced efficiently in a simple collision-induced dissociation-MS experiment. We applied the microarray approach to a detailed site-specific glycosylation analysis of human serum IgM. By scanning the treated spots with low-resolution matrix assisted laser desorption/ionization-time-of-flight-MS, we observed all five deglycosylated peptides, including the one originating from the secretory chain. A detailed glycopeptide characterization was then accomplished on the adjacent, untreated spots with high mass resolution and high mass accuracy using a matrix assisted laser desorption ionization-Fourier transform-MS. We present the first detailed and comprehensive mass spectrometric analysis on the glycopeptide level for human polyclonal IgM with high mass accuracy. Besides complex type glycans on Asn 395, 332, 171, and on the J chain, we observed oligomannosidic glycans on Asn 563, Asn 402 and minor amounts of oligomannosidic glycans on the glycosite Asn 171. Furthermore, hybrid type glycans were found on Asn 402, Asn 171 and in traces Asn 332.

A Microarray-Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry Approach for Site-specific Protein N-glycosylation Analysis, as Demonstrated for Human Serum Immunoglobulin M (IgM).

Hagfish slime is an ultra dilute, elastic and cohesive hydrogel that deploys within milliseconds in cold seawater from a glandularly secreted exudate. The slime is made of long keratin-like fibers and mucin-like glycoproteins that span a network which entraps water and acts as a defense mechanism against predators. Unlike other hydrogels, the slime only confines water physically and is very susceptible to mechanical stress, which makes it unsuitable for many processing operations and potential applications. Despite its huge potential, little work has been done to improve and functionalize the properties of this hydrogel. To address this shortcoming, hagfish exudate was mixed with a soy protein isolate suspension (4% w/v) and with a soy emulsion (commercial soy milk) to form a more stable structure and combine the functionalities of a suspension and emulsion with those of the hydrogel. Hagfish exudate interacted strongly with the soy systems, showing a markedly increased viscoelasticity and water retention. Hagfish mucin was found to induce a depletion and bridging mechanism, which caused the emulsion and suspension to flocculate, making "soy slime", a cohesive and cold-set emulsion- and particle gel. The flocculation network increases viscoelasticity and substantially contributes to liquid retention by entrapping liquid in the additional confinements between aggregated particles and protein fibers. Because the mucin-induced flocculation resembles the salt- or acid-induced flocculation in tofu curd production, the soy slime was cooked for comparison. The cooked soy slime was similar to conventional cooked tofu, but possessed a long-range cohesiveness from the fibers. The fibrous, cold-set, and curd-like structure of the soy slime represents a novel way for a cold coagulation and fiber incorporation into a suspension or emulsion. This mechanism could be used to efficiently gel functionalized emulsions or produce novel tofu-like structured food products.

Gelation of Soy Milk with Hagfish Exudate Creates a Flocculated and Fibrous Emulsion- and Particle Gel.

When hagfish (Myxinidae) are attacked by predators, they form a dilute, elastic, and cohesive defensive slime made of mucins and protein threads. In this study we propose a link between flow behavior and defense mechanism of hagfish slime. Oscillatory rheological measurements reveal that hagfish slime forms viscoelastic networks at low concentrations. Mucins alone did not contribute viscoelasticity, however in shear flow, viscosity was observed. The unidirectional flow, experienced by hagfish slime during suction feeding by predators, was mimicked with extensional rheology. Elongational stresses were found to increase mucin viscosity. The resulting higher resistance to flow could support clogging of the attacker's gills. Shear flow in contrast decreases the slime viscosity by mucin aggregation and leads to a collapse of the slime network. Hagfish may benefit from this collapse when trapped in their own slime and facing suffocation by tying a sliding knot with their body to shear off the slime. This removal could be facilitated by the apparent shear thinning behavior of the slime. Therefore hagfish slime, thickening in elongation and thinning in shear, presents a sophisticated natural high water content gel with flow properties that may be beneficial for both, defense and escape.

Hagfish slime and mucin flow properties and their implications for defense.

Bicelles composed of DMPC and phospholipids capable of chelating lanthanide ions, such as 1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine-diethylene triaminepentaacetate (DMPE-DTPA), are highly tunable magnetically responsive soft materials. Further doping of these systems with cholesterol-DTPA conjugates complexed to a lanthanide ion considerably enhances the bicelle's size and magnetic alignability. The high value of these cholesterol conjugates for bicelle design remains largely unexplored. Herein, we examine how molecular structural alterations within the cholesterol-DTPA conjugates lead to contrasting self-assembled polymolecular aggregate structures when incorporated into DMPC/DMPE-DTPA/Tm(3+) bilayers. The nature of the linker connecting the DTPA-chelating moiety to the sterol backbone is examined by synthesizing conjugates of various linker lengths and polarities. The incorporation of these compounds within the bilayer results in polymolecular aggregate geometries of higher curvature. The increasing degrees of freedom for conformational changes conveyed to the chelator headgroup with increasing linker atomic length reduce the cholesterol-DTPA conjugate's critical packing parameter. Consequently, an inverse correlation between the number of carbon atoms in the linker and the bicelle radius is established. The introduction of polarity into the carbon chain of the linker did not cause major changes in the polymolecular aggregate architecture. Under specific conditions, the additives permit the formation of remarkably temperature-resistant bicelles. The versatility of design offered by these amphiphiles gives rise to new and viable tools for the growing field of magnetically responsive soft materials.

Tailoring Bicelle Morphology and Thermal Stability with Lanthanide-Chelating Cholesterol Conjugates.

The magnetic susceptibility of lanthanide-chelating bicelles was selectively enhanced by introducing 3β-amino-5-cholestene (aminocholesterol, Chol-NH2) in the bilayer. Unprecedented magnetic alignment of the bicelles was achieved without altering their size. An aminocholesterol conjugate (Chol-C2OC2-NH2), in combination with different lanthanide ions, offers the possibility of fine-tuning the bicelle's magnetic susceptibility.

Mastering the magnetic susceptibility of magnetically responsive bicelles with 3β-amino-5-cholestene and complexed lanthanide ions.

Mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and its lanthanide ion (Ln3+) chelating phospholipid conjugate, 1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine-diethylene triaminepentaacetate (DMPE-DTPA), assemble into highly magnetically responsive polymolecular assemblies such as DMPC/DMPE-DTPA/Ln3+ (molar ratio 4:1:1) bicelles. Their geometry and magnetic alignability is enhanced by introducing cholesterol into the bilayer in DMPC/Cholesterol/DMPE-DTPA/Ln3+ (molar ratio 16:4:5:5). However, the reported fabrication procedures remain tedious and limit the generation of highly magnetically alignable species. Herein, a simplified procedure where freeze thawing cycles and extrusion are replaced by gentle heating and cooling cycles for the hydration of the dry lipid film was developed. Heating above the phase transition temperature Tm of the lipids composing the bilayer before cooling back below the Tm was essential to guarantee successful formation of the polymolecular assemblies composed of DMPC/DMPE-DTPA/Ln3+ (molar ratio 4:1:1). Planar polymolecular assemblies in the size range of hundreds of nanometers are achieved and deliver unprecedented gains in magnetic response. The proposed heating and cooling procedure further allowed to regenerate the highly magnetically alignable DMPC/Cholesterol/DMPE-DTPA/Ln3+ (molar ratio 16:4:5:5) species after storage for one month frozen at -18 °C. The simplicity and viability of the proposed fabrication procedure offers a new set of highly magnetically responsive lanthanide ion chelating phospholipid polymolecular assemblies as building blocks for the smart soft materials of tomorrow.

Methods for Generating Highly Magnetically Responsive Lanthanide-Chelating Phospholipid Polymolecular Assemblies.

Lanthanide ion (Ln3+) chelating amphiphiles are powerful molecules for tailoring the magnetic response of polymolecular assemblies. Mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine-diethylene triaminepentaacetate (DMPE-DTPA) complexed to Ln3+ deliver highly magnetically responsive bicelles. Their magnetic properties are readily tuned by changing the bicellar size or the magnetic susceptibility Δχ of the bilayer lipids. The former technique is intrinsically bound to the region of the phase diagram guarantying the formation of bicelles. Methods aiming towards manipulating the Δχ of the bilayer are comparatively more robust, flexible and lacking. Herein, we synthesized a new Ln3+ chelating phospholipid using glutamic acid as a backbone: DMPE-Glu-DTPA. The chelate polyhedron was specifically engineered to alter the Δχ, whilst remaining geometrically similar to DMPE-DTPA. Planar asymmetric assemblies hundreds of nanometers in size were achieved presenting unprecedented magnetic alignments. The DMPE-Glu-DTPA/Ln3+ complex switched the Δχ, achieving perpendicular alignment of assemblies containing Dy3+ and parallel alignment of those containing Tm3+. Moreover, samples with chelated Yb3+ were more alignable than the Tm3+ chelating counterparts. Such a possibility has never been demonstrated for planar Ln3+ chelating polymolecular assemblies. The physico-chemical properties of these novel assemblies were further studied by monitoring the alignment behavior at different temperatures and by including 16 mol% of cholesterol (Chol-OH) in the phospholipid bilayer. The DMPE-Glu-DTPA/Ln3+ complex and the resulting assemblies are promising candidates for applications in numerous fields including pharmaceutical technologies, structural characterization of membrane biomolecules by NMR spectroscopy, as contrasting agents for magnetic resonance imaging, and for the development of smart optical gels.

Molecular engineering of lanthanide ion chelating phospholipids generating assemblies with a switched magnetic susceptibility.

Cholesterol (Chol-OH) and its conjugates are powerful molecules for engineering the physicochemical and magnetic properties of phospholipid bilayers in bicelles. Introduction of aminocholesterol (3β-amino-5-cholestene, Chol-NH2) in bicelles composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and the thulium-ion-chelating phospholipid 1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine-diethylene triaminepentaacetate (DMPE-DTPA/Tm3+) results in unprecedented high magnetic alignments by selectively tuning the magnetic susceptibility Δχ of the bilayer. However, little is known on the underlying mechanisms behind the magnetic response and, more generally, on the physicochemical forces governing a Chol-NH2 doped DMPC bilayer. We tackled this shortcoming with a multiscale bottom-up comparative investigation of Chol-OH and Chol-NH2 mixed with DMPC. First, simplified monolayer models on a Langmuir trough were employed to compare the two steroid molecules at various contents in DMPC. In a second step, a molecular dynamics (MD) simulation allowed for a more representative model of the bicelle bilayer while monitoring the amphiphiles and their interactions on the molecular level. In a final step, we moved away from the models and investigated the effect of temperature on the structure and magnetic alignment of Chol-NH2 doped bicelles by SANS. The DMPC/steroid monolayer studies showed that Chol-OH induces a larger condensation effect than Chol-NH2 at steroid contents of 16 and 20 mol %. However, this tendency was inversed at steroid contents of 10, 30, and 40 mol %. Although the MD simulation with 16 mol % steroid revealed that both compounds induce a liquid-ordered state in DMPC, the bilayer containing Chol-NH2 was much less ordered than the analogous system containing Chol-OH. Chol-NH2 underwent significantly more hydrogen bonding interactions with neighboring DMPC lipids than Chol-OH. It seems that, by altering the dynamics of the hydrophilic environment of the bicelle, Chol-NH2 changes the crystal field and angle of the phospholipid-lanthanide DMPE-DTPA/Tm3+ complex. These parameters largely determine the magnetic susceptibility Δχ of the complex, explaining the SANS results, which show significant differences in magnetic alignment of the steroid doped bicelles. Highly magnetically alignable DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles were achieved up to temperatures of 35 °C before a thermoreversible rearrangement into nonalignable vesicles occurred. The results confirm the potential of Chol-NH2 doped bicelles to act as building blocks for the development of the magnetically responsive soft materials of tomorrow.

Understanding the Enhanced Magnetic Response of Aminocholesterol Doped Lanthanide-Ion-Chelating Phospholipid Bicelles.

Wormlike micellar aggregates formed from the mixture of ionic surfactants with aromatic additives result in solutions with impressive viscoelastic properties. These properties are of high interest for numerous industrial applications and are often used as model systems for soft matter physics. However, robust and simple models for tailoring the viscoelastic response of the solution based on the molecular structure of the employed additive are required to fully exploit the potential of these systems. We address this shortcoming with a modified packing parameter based model, considering the additive-surfactant pair. The role of charge neutralization on anisotropic micellar growth was investigated with derivatives of sodium salicylate. The impact of the additives on the morphology of the micellar aggregates is explained from the molecular level to the macroscopic viscoelasticity. Changes in the micelle's volume, headgroup area and additive structure are explored to redefine the packing parameter. Uncharged additives penetrated deeper into the hydrophobic region of the micelle, whilst charged additives remained trapped in the polar region, as revealed by a combination of 1H-NMR, SAXS and rheological measurements. A deeper penetration of the additives densified the hydrophobic core of the micelle and induced anisotropic growth by increasing the effective volume of the additive-surfactant pair. This phenomenon largely influenced the viscosity of the solutions. Partially penetrating additives reduced the electrostatic repulsions between surfactant headgroups and neighboring micelles. The resulting increased network density governed the elasticity of the solutions. Considering a packing parameter composed of the additive-surfactant pair proved to be a facile means of engineering the viscoelastic response of surfactant solutions. The self-assembly of the wormlike micellar aggregates could be tailored to desired morphologies resulting in a specific and predictable rheological response.

Simon Kuster

Laboratory of Food Process Engineering

ETH Zurich

Fabrication Procedures and Birefringence Measurements for Designing Magnetically Responsive Lanthanide Ion Chelating Phospholipid Assemblies

Simon Kuster

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory of Food Process Engineering

ETH Zurich

Fabrication Procedures and Birefringence Measurements for Designing Magnetically Responsive Lanthanide Ion Chelating Phospholipid Assemblies

Laboratory of Food Process Engineering