Multimode Analysis of Nanoscale Biomolecular Interactions

Biomolecular interactions, including protein-protein, protein-DNA, and protein-ligand interactions, are of special importance in all biological systems. These interactions may occer during the loading of biomolecules to interfaces, the translocation of biomolecules through transmembrane protein pores, and the movement of biomolecules in a crowded intracellular environment. The molecular interaction of a protein with its binding partners is crucial in fundamental biological processes such as electron transfer, intracellular signal transmission and regulation, neuroprotective mechanisms, and regulation of DNA topology. In this dissertation, a customized surface plasmon resonance (SPR) has been optimized and new theoretical and label free experimental methods with related analytical calculations have been developed for the analysis of biomolecular interactions. Human neuroglobin (hNgb) and cytochrome c from equine heart (Cyt c) proteins have been used to optimize the customized SPR instrument. The obtained Kd value (~13 µM), from SPR results, for Cyt c-hNgb molecular interactions is in general agreement with a previously published result. The SPR results also confirmed no significant impact of the internal disulfide bridge between Cys 46 and Cys 55 on hNgb binding to Cyt c. Using SPR, E. coli topoisomerase I enzyme turnover during plasmid DNA relaxation was found to be enhanced in the presence of Mg2+. In addition, a new theoretical approach of analyzing biphasic SPR data has been introduced based on analytical solutions of the biphasic rate equations. In order to develop a new label free method to quantitatively study protein-protein interactions, quartz nanopipettes were chemically modified. The derived Kd (~20 µM) value for the Cyt c-hNgb complex formations matched very well with SPR measurements (Kd ~16 µM). The finite element numerical simulation results were similar to the nanopipette experimental results. These results demonstrate that nanopipettes can potentially be used as a new class of a label-free analytical method to quantitatively characterize protein-protein interactions in attoliter sensing volumes, based on a charge sensing mechanism. Moreover, the molecule-based selective nature of hydrophobic and nanometer sized carbon nanotube (CNT) pores was observed. This result might be helpful to understand the selective nature of cellular transport through transmembrane protein pores.

Kunitzins: Prototypes of a new class of protease inhibitor from the skin secretions of European and Asian frogs

Amphibian skin secretions contain biologically-active compounds, such as anti-microbial peptides and trypsin inhibitors, which are used by biomedical researchers as a source of potential novel drug leads or pharmacological agents. Here, we report the application of a recently developed technique within our laboratory to “shotgun” clone the cDNAs encoding two novel but structurally-related peptides from the lyophilized skin secretions of one species of European frog, Rana esculenta and one species of Chinese frog, Odorrana schmackeri. Bioanalysis of the peptides established the structure of a 17-mer with an N-terminal Ala (A) residue and a C-terminal Cys (C) residue with a single disulphide bridge between Cys 12 and 17, which is a canonical Kunitz-type protease inhibitor motif (-CKAAFC-). Due to the presence of this structural attribute, these peptides were named kunitzin-RE (AAKIILNPKFRCKAAFC) and kunitzin-OS (AVNIPFKVHLRCKAAFC). Synthetic replicates of these two novel peptides were found to display a potent inhibitory activity against Escherichia coli but were ineffective at inhibiting the growth of Staphylococcus aureus and Candida albicans at concentrations up to 160 μM, and both showed little haemolytic activity at concentrations up to 120 μM. Subsequently, kunitzin-RE and kunitzin-OS were found to be a potent inhibitor of trypsin with a Ki of 5.56 μM and 7.56 μM that represent prototypes of a novel class of highly-attenuated amphibian skin protease inhibitor. Substitution of Lys-13, the predicted residue occupying the P1 position within the inhibitory loop, with Phe (F) resulted in decrease in trypsin inhibitor effectiveness and antimicrobial activity against Esherichia coli, but exhibits a potential inhibition activity against chymotrypsin.

Two new mu-(1,3-azido)-bridged polymers: alternating single and double bridges in a 1D nickel(II) complex and uniform bridge in a 2D copper(II) complex: Syntheses, single-crystal structures and magnetic studies

Two polymeric azido bridged complexes [Ni2L2(N-3)(3)](n)(ClO4). (1) and [Cu(bpdS)(2)(N-3)],(ClO4),(H2O)(2.5n) (2) [L = Schiff base, obtained from the condensation of pyridine-2-aldehyde with N,N,2,2-tetramethyl-1,3-propanediamine; bpds = 4,4'-bipyridyl disulfide] have been synthesized and their crystal structures have been determined. Complex 1, C26H42ClN15Ni2O4, crystallizes in a triclinic system, space group P1 with a 8.089(13), b = 9.392(14), c = 12.267(18) angstrom, a = 107.28(l), b 95.95(1), gamma = 96.92(1)degrees and Z = 2; complex 2, C20H21ClCuN7O6.5S4, crystallizes in an orthorhombic system, space group Pnna with a = 10.839(14), b = 13.208(17), c = 19.75(2) angstrom and Z = 4. The crystal structure of I consists of 1D polymers of nickel(L) units, alternatively connected by single and double bridging mu-(1,3-N-3) ligand with isolated perchlorate anions. Variable temperature magnetic susceptibility data of the complex have been measured and the fitting,of magnetic data was carried out applying the Borris-Almenar formula for such types of alternating one-dimensional S = 1 systems, based on the Hamiltonian H = -J Sigma(S2iS2i-1 + aS(2i)S(2i+1)). The best-fit parameters obtained are J = -106.7 +/- 2 cm(-1); a = 0.82 +/- 0.02; g = 2.21 +/- 0.02. Complex 2 is a 2D network of 4,4 topology with the nodes occupied by the Cu-II ions, and the edges formed by single azide and double bpds connectors. The perchlorate anions are located between pairs of bpds. The magnetic data have been fitted considering the complex as a pseudo-one-dimensional system, with all copper((II)) atoms linked by [mu(1,3-azido) bridging ligands at axial positions (long Cu...N-3 distances) since the coupling through long bpds is almost nil. The best-fit parameters obtained with this model are J = -1.21 +/- 0.2 cm(-1), g 2.14 +/- 0.02. (c) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005).

A plasmonic 'ac Wheatstone bridge' circuit for high-sensitivity phase measurement and single-molecule detection

In this paper, a plasmonic “ac Wheatstone bridge” circuit is proposed and theoretically modeled for the first time. The bridge circuit consists of three metallic nanoparticles, shaped as rectangular prisms, with two nanoparticles acting as parallel arms of a resonant circuit and the third bridging nanoparticle acting as an optical antenna providing an output signal. Polarized light excites localized surface plasmon resonances in the two arms of the circuit, which generate an optical signal dependent on the phase-sensitive excitations of surface plasmons in the antenna. The circuit is analyzed using a plasmonic coupling theory and numerical simulations. The analyses show that the plasmonic circuit is sensitive to phase shifts between the arms of the bridge and has the potential to detect the presence of single molecules.

Cyclic Peptide Disulfides - Solution And Solid-State Conformation Of Boc-Cys-Pro-Aib-Cys-S-S-Bridge-Nhme,A Disulfide-Bridged Peptide Helix

The solution and solid-state conformations of the peptide disulfide Boc-Cys-Pro-Aib-Cys-NHMe have been determined by NMR spectroscopy and X-ray diffraction. The Cys(4) and methylamide NH groups are solvent shielded in CDCI3 and (CD,),SO, suggesting their involvement in intramolecular hydrogen bonding. On the basis of known stereochemical preferences of Pro and Aib residues, a consecutive @-turn structure is favored in solution. X-ray diffraction analysis reveals a highly folded 310 helical conformation for the peptide, with the S-S bridge lying approximately parallel to the helix axis, linking residues 1 and 4. The backbone conformational angles are Cys(1) 4 = -121.1', $ = 65.6"; Pro(2) 4 = -58.9', 4 = -34.0'; Aib(3) 4 = -61.8', $ = -17.9'; Cys(4) 4 = -70.5', $ = -18.6'. Two intramolecular hydrogen bonds are observed between Cys(1) CO--HN Cys(4) and Pro(2) CO--HNMe. The disulfide bond has a right-handed chirality, with a dihedral angle (xss) of 82'.

Molecular structure of a cyclic tetrapeptide disulfide. A novel 310 helical conformation with an S-S bridge

The crystal structure of the cyclic peptide disulfide Boc-Cys-Pro-Aib-Cys-NHMe has been determined by X-ray diffraction. The peptide crystallizes in the space group P212121, with A = 8.646(1), B = 18.462(2), C = 19.678(3)Å and Z = 4. The molecules adopt a highly folded compact conformation, stabilized by two intramolecular 4→ 1 hydrogen bonds between the Cys (1) and Pro (2) CO groups and the Cys (4) and methylamide NH groups, respectively. The backbone conformational angles for the peptide lie very close to those expected for a 310 helix. The S-S bridge adopts a right handed twist with a dihedral angle of 82°. The structure illustrates the role of stereochemically constrained residues, in generating novel peptide conformations. Aib, α-aminoisobutyric acid; Z, benzyloxycarbonyl; Boc, t-butyloxycarbonyl; OMe, methyl ester; OBz, benzyl ester; NHMe, N-methylamide; Tosyl, p-toluenesulfonyl.

A simple analog controller for single-phase half-bridge rectifier and its application to transformerless UPS

A simple, low-cost, constant frequency, analog controller is proposed for the front-end half-bridge rectifier of a single-phase transformerless UPS system to maintain near unity power factor at the input and zero dc-offset voltage at the output. The controller generates the required gating pulses by comparing the input current with a periodic, bipolar, linear carrier without sensing the input voltage. Two voltage controllers and a single integrator with reset are used to generate the required carrier. All the necessary control operations can be performed without using any PLL, multiplier and/or divider. The controller can be fabricated as a single integrated circuit. The control concept is validated through simulation and also experimentally on an 800W half-bridge rectifier. Experimental results are presented for ac-dc application, and also for ac-dc-ac UPS application with both sinusoidal and nonlinear loads. The simulation and experimental results agree well.

A Five-Level Inverter Topology with Single-DC Supply by Cascading a Flying Capacitor Inverter and an H-Bridge

In this paper, a new three-phase, five-level inverter topology with a single-dc source is presented. The proposed topology is obtained by cascading a three-level flying capacitor inverter with a flying H-bridge power cell in each phase. This topology has redundant switching states for generating different pole voltages. By selecting appropriate switching states, the capacitor voltages can be balanced instantaneously (as compared to the fundamental) in any direction of the current, irrespective of the load power factor. Another important feature of this topology is that if any H-bridge fails, it can be bypassed and the configuration can still operate as a three-level inverter at its full power rating. This feature improves the reliability of the circuit. A 3-kW induction motor is run with the proposed topology for the full modulation range. The effectiveness of the capacitor balancing algorithm is tested for the full range of speed and during the sudden acceleration of the motor.

A Three-Level Common-Mode Voltage Eliminated Inverter With Single DC Supply Using Flying Capacitor Inverter and Cascaded H-Bridge

A three-level common-mode voltage eliminated inverter with single dc supply using flying capacitor inverter and cascaded H-bridge has been proposed in this paper. The three phase space vector polygon formed by this configuration and the polygon formed by the common-mode eliminated states have been discussed. The entire system is simulated in Simulink and the results are experimentally verified. This system has an advantage that if one of devices in the H-bridge fails, the system can still be operated as a normal three-level inverter at full power. This inverter has many other advantages like use of single dc supply, making it possible for a back-to-back grid-tied converter application, improved reliability, etc.

YBCO single grains seeded by 45° - 45° bridge-seeds of different lengths

Single grain, (RE)BCO bulk superconductors in large or complicated geometries are required for a variety of potential applications, such as motors and generators and magnetic shielding devices. As a result, top, multi-seeded, melt growth (TMSMG) has been investigated over the past two years in an attempt to enlarge the size of (RE)BCO single grains specifically for such applications. Of these multi-seeding techniques, so-called bridge seeding provides the best alignment of two seeds in a single grain growth process. Here we report, for the first time, the successful growth of YBCO using a special, 45{\deg} - 45{\deg}, arrangement of bridge-seeds. The superconducting properties, including trapped field, of the multi-seeded YBCO grains have been measured for different bridge lengths of the 45{\deg}- 45{\deg} bridge-seeds. The boundaries at the impinging growth front and the growth features of the top, multi-seeded surface and cross-section of the multi-seeded, samples have been analysed using optical microscopy. The results suggest that an impurity-free boundary between the two seeds of each leg of the bridge-seed can form when 45{\deg}- 45{\deg} bridge-seeds are used to enlarge the size of YBCO grains.

A comparison of 0°-0° and 45°-45° bridge-seeded, YBCO single grains

A variety of multiseeding techniques have been investigated over the past 20 yr in an attempt to enlarge bulk (RE)BCO superconducting samples fabricated by the top-seeded melt growth (TSMG) process for practical applications. Unfortunately, these studies have failed to establish whether technically useful values of trapped field can be achieved in multiseeded bulk samples. In this work specially designed, 0°-0° and 45°-45° bridge seeds of different lengths have been employed to produce improved alignment of the seeds during the TSMG process. The ability of these bridge-seeded samples to trap magnetic field, which is the key superconducting property for practical applications of bulk (RE)BCO, is compared for the samples seeded using 0°-0° and 45°-45° bridge seeds of different lengths. The grain boundaries produced by these bridge seeds are analyzed in detail, and the similarities and differences between the two bridge-seeding processes are discussed. © 2013 The American Ceramic Society.

Analysis and retrofitting design of a single-span R.C. bridge

This paper deals with the case history of a damaged one-span prestressed concrete bridge on a crucial artery near the city of Cagliari (Sardinia), along the sea-side. After being involved in a disastrous flood, attention has arisen on the worrying safety state of the deck, submitted to an intense daily traffic load. Evident signs of this severe condition were the deterioration of the beams concrete and the corrosion, the lack of tension and even the rupture of the prestressing cables. After performing a limited in situ test campaign, consisting of sclerometer, pull out and carbonation depth tests, a first evaluation of the safety of the structure was performed. After collecting the data of dynamic and static load tests as well, a comprehensive analysis have been carried out, also by means of a properly calibrated F.E. model. Finally the retrofitting design is presented, consisting of the reparation and thickening of the concrete cover, providing flexural and shear FRP external reinforcements and an external prestressing system, capable of restoring a satisfactory bearing capacity, according to the current national codes. The intervention has been calibrated by the former F.E. model with respect to transversal effects and influence of local and overall deformation of reinforced elements. © 2012 Taylor & Francis Group.