Impact of A134 and E218 Amino Acid Residues of Tropomyosin on Its Flexibility and Function

Tropomyosin (Tpm) is one of the major actin-binding proteins that play a crucial role in the regulation of muscle contraction. The flexibility of the Tpm molecule is believed to be vital for its functioning, although its role and significance are under discussion. We choose two sites of the Tpm molecule that presumably have high flexibility and stabilized them with the A134L or E218L substitutions. Applying differential scanning calorimetry (DSC), molecular dynamics (MD), co-sedimentation, trypsin digestion, and in vitro motility assay, we characterized the properties of Tpm molecules with these substitutions. The A134L mutation prevented proteolysis of Tpm molecule by trypsin, and both substitutions increased the thermal stability of Tpm and its bending stiffness estimated from MD simulation. None of these mutations affected the primary binding of Tpm to F-actin; still, both of them increased the thermal stability of the actin-Tpm complex and maximal sliding velocity of regulated thin filaments in vitro at a saturating Ca²⁺ concentration. However, the mutations differently affected the Ca²⁺ sensitivity of the sliding velocity and pulling force produced by myosin heads. The data suggest that both regions of instability are essential for correct regulation and fine-tuning of Ca²⁺-dependent interaction of myosin heads with F-actin.     

Mass spectrometry and the cellular surfaceome

The collection of exposed plasma membrane proteins, collectively termed the surfaceome, is involved in multiple vital cellular processes, such as the communication of cells with their surroundings and the regulation of transport across the lipid bilayer. The surfaceome also plays key roles in the immune system by recognizing and presenting antigens, with its possible malfunctioning linked to disease. Surface proteins have long been explored as potential cell markers, disease biomarkers, and therapeutic drug targets. Despite its importance, a detailed study of the surfaceome continues to pose major challenges for mass spectrometry-driven proteomics due to the inherent biophysical characteristics of surface proteins. Their inefficient extraction from hydrophobic membranes to an aqueous medium and their lower abundance compared to intracellular proteins hamper the analysis of surface proteins, which are therefore usually underrepresented in proteomic datasets. To tackle such problems, several innovative analytical methodologies have been developed. This review aims at providing an extensive overview of the different methods for surfaceome analysis, with respective considerations for downstream mass spectrometry-based proteomics.     

Determination of microstructural characteristics of tungsten nanopowders from the profile of a single diffraction peak using WinFit software

WinFit software is used to determine using the profile of a single diffraction peak the sizes of coherent scattering domains and microstrains for tungsten powders obtained through plasma chemical synthesis and hydrogen reduction from tungstic acid. It is shown that the use of the (110), (200) or (211) diffraction peaks is the most expedient for the calculations. At the same time, it should be noted that, in the case of significant microstrains, the sizes of coherent scattering domains calculated using the second and the third peaks are underestimated.     

Effect of electrostatic shielding on the photoelectric properties of heterostructures with deep QWs

The effect of the electrostatic potential induced by charge carriers of the same sign, localized in a deep quantum well, on the current–voltage characteristics of photodetector heterostructures is theoretically analyzed. It is shown for the example of a p–i–n structure with a single deep quantum well in the i-type region that the shielding of an external electric field makes the differential photoconductivity of the heterostructure higher than that in a p–i–n structure without an intermediate 2D layer.     

SEM-EDX analysis in the source apportionment of particulate matter on Hypogymnia physodes lichen transplants around the Cu smelter and former mining town of Karabash, South Urals, Russia

Scanning electron microscopy with energy-dispersive X-ray analysis (SEM-EDX) of particulate matter on lichen transplant thalli (Hypogymnia physodes) was assessed as a complementary technique to wet chemical analysis for source apportionment of airborne contaminants. Transplants (2 month exposure) stationed in the Cu smelter and former mining town of Karabash were compared with those from a control site 30 km south. Particulate matter in Karabash samples (715 analyses) showed higher levels of S, Pb, Cu, Sn and Zn compared with the control (598 analyses). Complex element associations among the particles confounded detailed mineralogical identifications, and therefore a simplified particle classification scheme was devised for source apportionment. Karabash samples contained high levels of particles classified as mining-related (MRP), and these were also identified in control samples, indicating wide spatial dispersion from the smelter and highlighting the sensitivity of the method. It was noted that MRP <2.5-microm diameter were poorly represented on lichen surfaces suggesting this may limit the usefulness of Hypogymnia transplants as proxies when assessing human health impacts from airborne particulates. Analyses of the lichen thallus surface (away from surface particulates) revealed high levels of Cu, Zn, Fe and Pb associated with organics in the Karabash samples compared with the control, with a proportionate loss of K, interpreted as being due to a stress-related increase in cell membrane permeability. This type of analysis may provide a novel SEM-EDX-based method for assessing lichen vitality. The techniques developed are presented and further implications of the study are discussed.     

Sensor properties of hollow-core photonic crystal fibers

A new class of hollow-core photonic crystal fibers (PCFs) with various modifications of spatial lattices is considered. The influence of the PCF structure geometry on the photonic band gap (PBG) in the visible spectral range is demonstrated. A relationship between a change in the PCF core refractive index due to filling of the hollow core with various liquids and the PBG position is established. PCFs show new possibilities for use as biological and chemical sensors.     

Holographic projection of arbitrary light patterns with a suppressed zero-order beam

We present what is to our knowledge a novel technique for efficient suppression of the zero-order beam inherent in light patterns projected via phase-only computer-generated holograms (CGHs). Encoding a CGH on a spatial light modulator (SLM) with a limited fill factor produces a disturbing zero-order beam at the optical axis. Here, we propose to derive a CGH, which includes holographic information to project a corrective beam that destructively interferes with the zero-order beam. The CGH for projecting arbitrary light patterns plus a corrective beam are derived using the Gerchberg-Saxton algorithm where the iterations impose both amplitude and phase constraints for the target field pattern at the Fourier plane. As proof of principle, we analyze the viability of the technique by simulating the performance when applied on a practical SLM with a limited fill factor, fixed number of phase-shifting pixels, and wavefront distortion associated with the surface roughness of the SLM.     

Performance enhancement in high-<Emphasis Type="Italic">T</Emphasis><Subscript><Emphasis Type="Italic">c</Emphasis></Subscript> ceramics through melting by Joule heating,infrared lamps,and laser radiation

We have analyzed experimental data on the effect of short-term melting followed by recrystallization on the microstructure and critical current density of YBa₂Cu₃O_{7 ? x} , Bi₂Sr₂CaCu₂O_{8 ? x} , and Bi₂Sr₂Ca₂Cu₃O_{10 ? x} high-T _c ceramics. The ceramics were melted using different heat sources: infrared lamps, laser radiation, and electric current. A significant increase in the critical current density of Bi₂Sr₂Ca₂Cu₃O_{10 ? x} ceramics (by a factor of 40 at 20 K and by a factor of 8 at 77 K) was achieved using cw CO₂ laser irradiation. Melting TiC-doped (0.1%) Bi₂Sr₂Ca₂Cu₃O_{10 ? x} ceramics with a CO₂ laser, followed by annealing, insured an even larger increase in critical current density: by a factor of 35 at 77 K. We have calculated the thickness of the molten layer produced by laser heating of high-T _c ceramics.     

Esterase Activity of Serum Albumin Studied by 1H NMR Spectroscopy and Molecular Modelling

Serum albumin possesses esterase and pseudo-esterase activities towards a number of endogenous and exogenous substrates, but the mechanism of interaction of various esters and other compounds with albumin is still unclear. In the present study, proton nuclear magnetic resonance (¹H NMR) has been applied to the study of true esterase activity of albumin, using the example of bovine serum albumin (BSA) and p-nitrophenyl acetate (NPA). The site of BSA esterase activity was then determined using molecular modelling methods. According to the data obtained, the accumulation of acetate in the presence of BSA in the reaction mixture is much more intense as compared with the spontaneous hydrolysis of NPA, which indicates true esterase activity of albumin towards NPA. Similar results were obtained for p-nitophenyl propionate (NPP) as substrate. The rate of acetate and propionate release confirms the assumption that there is a site of true esterase activity in the albumin molecule, which is different from the site of the pseudo-esterase activity Sudlow II. The results of molecular modelling of BSA and NPA interaction make it possible to postulate that Sudlow site I is the site of true esterase activity of albumin.     

Identification and Affinity Determination of Protein-Antibody and Protein-Aptamer Epitopes by Biosensor-Mass Spectrometry Combination

Analytical methods for molecular characterization of diagnostic or therapeutic targets have recently gained high interest. This review summarizes the combination of mass spectrometry and surface plasmon resonance (SPR) biosensor analysis for identification and affinity determination of protein interactions with antibodies and DNA-aptamers. The binding constant (K_D) of a protein–antibody complex is first determined by immobilizing an antibody or DNA-aptamer on an SPR chip. A proteolytic peptide mixture is then applied to the chip, and following removal of unbound material by washing, the epitope(s) peptide(s) are eluted and identified by MALDI-MS. The SPR-MS combination was applied to a wide range of affinity pairs. Distinct epitope peptides were identified for the cardiac biomarker myoglobin (MG) both from monoclonal and polyclonal antibodies, and binding constants determined for equine and human MG provided molecular assessment of cross immunoreactivities. Mass spectrometric epitope identifications were obtained for linear, as well as for assembled (“conformational”) antibody epitopes, e.g., for the polypeptide chemokine Interleukin-8. Immobilization using protein G substantially improved surface fixation and antibody stabilities for epitope identification and affinity determination. Moreover, epitopes were successfully determined for polyclonal antibodies from biological material, such as from patient antisera upon enzyme replacement therapy of lysosomal diseases. The SPR-MS combination was also successfully applied to identify linear and assembled epitopes for DNA–aptamer interaction complexes of the tumor diagnostic protein C-Met. In summary, the SPR-MS combination has been established as a powerful molecular tool for identification of protein interaction epitopes.