Tracking Conformational Changes in Calmodulin in vitro,in Cell Extract,and in Cells by Electron Paramagnetic Resonance Distance Measurements

It is an open question whether the conformations of proteins sampled in dilute solutions are the same as in the cellular environment. Here we address this question by double electron-electron resonance (DEER) distance measurements with Gd(III) spin labels to probe the conformations of calmodulin (CaM) in vitro, in cell extract, and in human HeLa cells. Using the CaM mutants N53C/T110C and T34C/T117C labeled with maleimide-DOTA-Gd(III) in the N- and C-terminal domains, we observed broad and varied interdomain distance distributions. The in vitro distance distributions of apo-CaM and holo-CaM in the presence and absence of the IQ target peptide can be described by combinations of closed, open, and collapsed conformations. In cell extract, apo- and holo-CaM bind to target proteins in a similar way as apo- and holo-CaM bind to IQ peptide in vitro. In HeLa cells, however, in the presence or absence of elevated in-cell Ca²⁺ levels CaM unexpectedly produced more open conformations and very broad distance distributions indicative of many different interactions with in-cell components. These results show-case the importance of in-cell analyses of protein structures.     

Spin‐Labeled Heparins as Polarizing Agents for Dynamic Nuclear Polarization

A potentially biocompatible class of spin‐labeled macromolecules, spin‐labeled (SL) heparins, and their use as nuclear magnetic resonance (NMR) signal enhancers are introduced. The signal enhancement is achieved through Overhauser‐type dynamic nuclear polarization (DNP). All presented SL‐heparins show high ¹H DNP enhancement factors up to E=?110, which validates that effectively more than one hyperfine line can be saturated even for spin‐labeled polarizing agents. The parameters for the Overhauser‐type DNP are determined and discussed. A striking result is that for spin‐labeled heparins, the off‐resonant electron paramagnetic resonance (EPR) hyperfine lines contribute a non‐negligible part to the total saturation, even in the absence of Heisenberg spin exchange (HSE) and electron spin‐nuclear spin relaxation (T_1ne). As a result, we conclude that one can optimize the use of, for example, biomacromolecules for DNP, for which only small sample amounts are available, by using heterogeneously distributed radicals attached to the molecule.     

Interaction of an Endosomolytic Polyamidoamine ISA23 with Vesicles Mimicking Intracellular Membranes: A SANS/EPR Study

The mechanism of ISA23 · HCl interaction with model membrane vesicles (80–100 nm in diameter) was investigated using EPR in conjunction with SANS. For EPR, 16‐DSE was dissolved in the vesicle membrane to measure its dynamics and polarity, whereas a spin‐labeled (Tempo)‐ISA 23 analogue was used to give a measure of the polymer flexibility. When ISA23 was added to the external vesicle surface, no interaction was found. This observation conflicts with the reported ability to lyse RBC, but is in agreement with recent studies that showed no effect on membrane permeability when a PAA was added to an incubation medium containing isolated lysosomal vesicles. The vesicle‐mimetic models used here provide a new and useful tool for studying endosomolytic polymer/membrane interactions.

     

Performance of Fluorescent Labels in Sedimentation Bead Arrays—A Comparison Study

An extensive study is described to identify the most suitable fluorescent label in magnetic microsphere sedimentation arrays. The investigated fluorescent labels, commonly used in multiplex analysis, include organic dyes, (fluorescein, Alexa488, Cy5) fluorescent proteins (R-Phycoerythrin, Allophycocyanine, PBXL-3) polymer nanoparticles (FluoSpheres, PD-Pt) and semiconductor nanocrystals (Quantum dots). DNA hybridization assays on magnetic microspheres were applied as model systems to reveal label performance. The fluorescent labels were characterized under optimized conditions regarding signal intensity, non-specific binding and photo-stability. The advantages and drawbacks of individual labels are discussed. The limit of detection and dynamic ranges are determined to compare the performance of selected labels. Detection limits of 2 × 10⁻¹⁰ mol/L are found for the determination of oligonucleotides using PBXl-3 as label, which is comparable with typical flow cytometer systems. The results and protocols are highly valuable for any type of bead based assays and can be easily transferred.     

Modification of Poly‐ and Oligosaccharides with Os(VI)pyridine. Voltammetry of the Os(VI) Adducts Obtained by Ligand Exchange

Polysaccharides and oligosaccharides were modified with Os(VI)pyridine complex followed by ligand exchange with different ligands such as 2,2′‐bipyridine or N,N,N′,N′‐tetramethylethylenediamine. The time of the modification was much shorter (taking about 15 min) then direct modification with the given Os(VI) complex. The resulting saccharide adducts were analyzed by voltammetric methods at carbon and mercury electrodes. The results showed that the proposed technique gives promise for a new approach to analysis of glycoproteins.     

Analysis of close proximity quenching of phosphorescent metalloporphyrin labels in oligonucleotide structures

Quenching of phosphorescent platinum(II) and palladium(II) coproporphyrin (MeCP) labelled oligonucleotides was investigated. Strong hybridization-specific quenching was observed in duplex DNA structures with a variety of quenchers and with two identical porphyrin labels when in close proximity. Classical resonance energy transfer mechanism was ruled out, since quenching did not correlate with spectral overlaps and lifetime changes were insignificant. Quenching of MeCP by the free quenchers in solution revealed that porphyrin-porphyrin quenching is predominantly static while other dyes quench dynamically. The results suggest that the quenching in DNA duplex proceeds via direct contact.     

Combination of Mass Signal Amplification and Isotope‐Labeled Alkanethiols for the Multiplexed Detection of miRNAs

We report a fast and sensitive method for the multiplexed detection of miRNAs by combining mass signal amplification and isotope‐labeled signal reporter molecules. In our strategy, target miRNAs are captured specifically by immobilized DNAs on gold nanoparticles (AuNPs), which carry a large number of small molecules, called amplification tags (Am‐tags), as the reporter for the detection of target miRNAs. For multiplexed detection, we designed and synthesized four Am‐tags containing 0, 4, 8, 12 isotopes so that they had same molecular properties but different molecular weights. By observing the mass signals of the Am‐tags on AuNPs decorated along with different probe DNAs, four types of miRNAs in a sample could be easily discriminated, and the relative amounts of these miRNAs could be quantified. The practicability of our strategy was further verified by measuring the expression levels of two miRNAs in HUVECs in response to different CuSO₄ concentrations.     

A Reactive,Rigid GdIII Labeling Tag for In‐Cell EPR Distance Measurements in Proteins

The cellular environment of proteins differs considerably from in vitro conditions under which most studies of protein structures are carried out. Therefore, there is a growing interest in determining dynamics and structures of proteins in the cell. A key factor for in‐cell distance measurements by the double electron–electron resonance (DEER) method in proteins is the nature of the used spin label. Here we present a newly designed Gd^III spin label, a thiol‐specific DOTA‐derivative (DO3MA‐3BrPy), which features chemical stability and kinetic inertness, high efficiency in protein labelling, a short rigid tether, as well as favorable spectroscopic properties, all are particularly suitable for in‐cell distance measurements by the DEER method carried out at W‐band frequencies. The high performance of DO3MA‐3BrPy‐Gd^III is demonstrated on doubly labelled ubiquitin D39C/E64C, both in vitro and in HeLa cells. High‐quality DEER data could be obtained in HeLa cells up to 12 h after protein delivery at in‐cell protein concentrations as low as 5–10 μm .     

Defects,flexoelectricity and RF communications: the ZBD story

ABSTRACT

The zenithal bistable display (ZBD) was the first liquid crystal device mode to be commercialised that uses nematic disclinations in a constructive fashion, to use the flexoelectric effect inherent to all liquid crystals but at the time was considered too weak an effect to be useful, and to transfer nano-replication methods to the LCD manufacturing environment. The genesis of the invention and spin-out company ZBD Displays Limited will be described, and the evolution of that company from licensing model, through fabless manufacturer to display provider and finally to a system provider for the retail sector. The story may be useful not just to those interested in the science behind a rather unusual LCD, but also those involved in taking technology from laboratory to manufacturing, from idea to commercial success.