Plasmon-enhanced refractometry using silver nanowire coatings on tilted fibre Bragg gratings.

A novel technique for increasing the sensitivity of tilted fibre Bragg grating (TFBG) based refractometers is presented. The TFBG sensor was coated with chemically synthesized silver nanowires ~100 nm in diameter and several micrometres in length. A 3.5-fold increase in sensor sensitivity was obtained relative to the uncoated TFBG sensor. This increase is associated with the excitation of surface plasmons by orthogonally polarized fibre cladding modes at wavelengths near 1.5 µm. Refractometric information is extracted from the sensor via the strong polarization dependence of the grating resonances using a Jones matrix analysis of the transmission spectrum of the fibre.

Self-optimized metal coatings for fiber plasmonics by electroless deposition.

We present a novel method to prepare optimized metal coatings for infrared Surface Plasmon Resonance (SPR) sensors by electroless plating. We show that Tilted Fiber Bragg grating sensors can be used to monitor in real-time the growth of gold nano-films up to 70 nm in thickness and to stop the deposition of the gold at a thickness that maximizes the SPR (near 55 nm for sensors operating in the near infrared at wavelengths around 1550 nm). The deposited films are highly uniform around the fiber circumference and in spite of some nanoscale roughness (RMS surface roughness of 5.17 nm) the underlying gratings show high quality SPR responses in water.

Near-field scanning optical microscopy probes: a comparison of pulled and double-etched bent NSOM probes for fluorescence imaging of biological samples.

Bent near-field optical probes for biological applications have been fabricated using a combination of a two-step chemical etching method and focused ion beam milling to create a well-defined aperture. The transmission efficiencies have been evaluated as a function of laser wavelength (lambda) and aperture size (D) for both large and small core fibres. The probe transmission behaviour follows a (D/lambda)3 relationship. The double-etched probes are compared to pulled probes fabricated from highly GeO2-doped dispersion compensating fibre and a standard single-mode optical fibre. The transmission efficiencies of both types of pulled probes are approximately two orders of magnitude lower than double-etched probes with similar aperture sizes. To demonstrate the utility of the various probes, their imaging performance has been evaluated for samples of polymer beads and phase-separated phospholipid monolayers of dipalmitoylphosphatidylcholine or cholesterol/phosphatidylcholine/sphingomyelin mixtures. Both pulled and double-etched probes are suitable for fluorescence imaging of polymer spheres. However, pulled probes are rapidly damaged at the higher input laser intensities required for fluorescence imaging of monolayer samples doped with < 1% of a fluorescent dye-labelled lipid. The images obtained with the double-etched probes show excellent spatial resolution and signal/noise, illustrating the potential of such probes for imaging of biological samples.

Dihedral psi angle dependence of the amide III vibration: a uniquely sensitive UV resonance Raman secondary structural probe.

UV resonance Raman studies of peptide and protein secondary structure demonstrate an extraordinary sensitivity of the amide III (Am III) vibration and the C(alpha)H bending vibration to the amide backbone conformation. We demonstrate that this sensitivity results from a Ramachandran dihedral psi angle dependent coupling of the amide N-H motion to (C)C(alpha)H motion, which results in a psi dependent mixing of the Am III and the (C)C(alpha)H bending motions. The vibrations are intimately mixed at psi approximately 120 degrees, which is associated with both the beta-sheet conformation and random coil conformations. In contrast, these motions are essentially unmixed for the alpha-helix conformation where psi approximately -60 degrees. Theoretical calculations demonstrate a sinusoidal dependence of this mixing on the psi angle and a linear dependence on the distance separating the N-H and (C)C(alpha)H hydrogens. Our results explain the Am III frequency dependence on conformation as well as the resonance Raman enhancement mechanism for the (C)C(alpha)H bending UV Raman band. These results may in the future help us extract amide psi angles from measured UV resonance Raman spectra.

Molecular determinants of site-specific inhibition of human DNA topoisomerase I by fagaronine and ethoxidine. Relation to DNA binding.

DNA topoisomerase (top) I inhibition activity of the natural alkaloid fagaronine (NSC157995) and its new synthetic derivative ethoxidine (12-ethoxy-benzo[c]phenanthridine) has been correlated with their molecular interactions and sequence specificity within the DNA complexes. Flow linear dichroism shows that ethoxidine exhibits the same inhibition of DNA relaxation as fagaronine at the 10-fold lower concentration. The patterns of DNA cleavage by top I show linear enhancement of CPT-dependent sites at the 0.016-50 microM concentrations of fagaronine, whereas ethoxidine suppress both top I-specific and CPT-dependent sites. Suppression of top I-mediated cleavage by ethoxidine is found to be specific for the sites, including strand cut between A and T. Fagaronine and ethoxidine are DNA major groove intercalators. Ethoxidine intercalates DNA in A-T sequences and its 12-ethoxy-moiety (absent in fagaronine) extends into the DNA minor groove. These findings may explain specificity of suppression by ethoxidine of the strong top I cleavage sites with the A(+1), T(-1) immediately adjacent to the strand cut. Fagaronine does not show any sequence specificity of DNA intercalation, but its highly electronegative oxygen of hydroxy group (absent in ethoxidine) is shown to be an acceptor of the hydrogen bond with the NH(2) group of G base of DNA. Ability of fagaronine to stabilize top I-mediated ternary complex is proposed to be determined by interaction of its hydroxy group with the guanine at position (+1) of the DNA cleavage site and of quaternary nitrogen interaction with top I. The model proposed provides a guidance for screening new top I-targeted drugs in terms of identification of molecular determinants responsible for their top I inhibition effects.

Expression, purification and DNA-cleavage activity of recombinant 68-kDa human topoisomerase I-target for antitumor drugs.

The gene encoding human DNA topoisomerase (topo) I, the target of numerous anticancer drugs, has been subcloned into bacterial, yeast and baculovirus-based expression systems in attempts to overexpress the enzyme for extensive structural and functional characterisation. Expression in E.coli produced a protein which was not suitable for structural studies. Expression in the yeast system was more successful enabling the enzyme to be purified and characterised. However, the resulting yield was modest for our requirements and the full-length protein was found to be susceptible to proteolysis when expressed in this system. As it is known that topo I from human placental tissue contains significant quantities of a 68kDa proteolytic fragment which retains both DNA relaxation and cleavage activity, we have isolated this fragment and shown by N-terminal sequence analysis that it starts at Lysine-191. This information was used to construct vectors which direct the overexpression of this fragment in baculovirus infected insect cells. The recombinant protein has been purified to homogeneity in a yield of 5-10mg/l of cell culture. The fragment is stable and retains all of the DNA driving activities of the intact enzyme. We have characterised the interactions of the topo I fragment with synthetic DNA substrates and identified oligonucleotides and conditions that allow covalent complexes between 68kDa topo I and DNA to be formed with high efficiency and in large quantity. A flow linear dichroism technique has been further developed and applied for real-time monitoring of supercoiled (sc) DNA relaxation by the enzyme and for comparative analysis of inhibition of 68kDa topo I by camptothecin (CPT).

Raman and CD spectroscopy of recombinant 68-kDa DNA human topoisomerase I and its complex with suicide DNA-substrate.

N-terminally truncated recombinant 68-kDa human topoisomerase (topo) I exhibits the same DNA-driving activities as the wild-type protein. In the present study, Raman and circular dichroism techniques were employed for detailed structural characterization of the 68-kDa human topo I and its transformations induced by the suicide sequence-specific oligonucleotide (solig) binding and cleavage. Spectroscopic data combined with statistical prediction techniques were employed to construct a model of the secondary structure distribution along the primary protein structure in solution. The 68-kDa topo I was found to consist of ca. 59% alpha-helix, 24% beta-strand and/or sheets, and 17% other structures. A secondary structure transition of the 68-kDa topo I was found to accompany solig binding and cleavage. Nearly 15% of the alpha-helix of 68-kDa topo I is transferred within the other structures when in the complex with its DNA substrate. Raman spectroscopy analysis also shows redistribution of the structural rotamers of the 68-kDa topo I disulfide bonds and significant changes in the H-bonding of the Tyr residues and in the microenvironment/conformation of the Trp side chains. No structural modifications of the DNA substrate were detected by spectroscopic techniques. The data presented provide the first direct experimental evidence of the human topo I conformational transition after the cleavage step in the reaction of binding and cleavage of DNA substrate by the enzyme. This evidence supports the model of the enzyme function requiring the protein conformational transition. The most probable location of the enzyme transformations was the core and the C-terminal conservative 68-kDa topo I structural domains. By contrast, the linker domain was found to have an extremely low potential for solig-induced structural transformations. The pattern of redistribution of protein secondary structures induced by solig binding and covalent suicide complex formation supports the model of an intramolecular bipartite mode of topo I/DNA interaction in the substrate binding and cleavage reaction.

Camptothecin-binding site in human serum albumin and protein transformations induced by drug binding.

Circular dichroism (CD) and Raman spectroscopy were employed in order to locate a camptothecin (CPT)-binding site within human serum albumin (HSA) and to identify protein structural transformations induced by CPT binding. A competitive binding of CPT and 3'-azido-3'-deoxythymidine (a ligand occupying IIIA structural sub-domain of the protein) to HSA does not show any competition and demonstrates that the ligands are located in the different binding sites, whereas a HSA-bound CPT may be replaced by warfarin, occupying IIA structural sub-domain of the protein. Raman and CD spectra of HSA and HSA/CPT complexes show that the CPT-binding does not induce changes of the global protein secondary structure. On the other hand, Raman spectra reveal pronounced CPT-induced local structural modifications of the HSA molecule, involving changes in configuration of the two disulfide bonds and transfer of a single Trp-residue to hydrophilic environment. These data suggest that CPT is bound in the region of interdomain connections within the IIA structural domain of HSA and it induces relative movement of the protein structural domains.