Toward an Alternative Intrinsic Probe for Spectroscopic Characterization of a Protein

The intrinsic fluorescent amino acid tryptophan is the unanimous choice for the spectroscopic investigation of proteins. However, several complicacies in the interpretation of tryptophan fluorescence in a protein are inevitable and an alternative intrinsic protein probe is a longstanding demand. In this contribution, we report an electron-transfer reaction in a human transporter protein (HSA) cavity which causes the tryptophan residue (Trp214) to undergo chemical modification to form one of its metabolites kynurenine (Kyn214). Structural integrity upon modification of the native protein is confirmed by dynamic light scattering (DLS) as well as near and far circular dichroism (CD) spectroscopy. Femtosecond-resolved fluorescence transients of the modified protein describe the dynamics of solvent molecules in the protein cavity in both the native and denatured states. In order to establish general use of the probe, we have studied the dipolar interaction of Kyn214 with a surface-bound ligand (crystal violet, CV) of the protein. By using the sensitivity of FRET, we have determined the distance between Kyn214 (donor) and CV (acceptor). Our study is an attempt to explore an alternative intrinsic fluorescence probe for the spectroscopic investigation of a protein. In order to establish the efficacy of the modification technique we have converted the tryptophan residues of other proteins (bovine serum albumin, chymotrypsin and subtilisin Carlsberg) to kynurenine and confirmed their structural integrity. We have also shown that catalytic activity of the enzymes remains intact upon the modification

Interaction of an Antituberculosis Drug with a Nanoscopic Macromolecular Assembly: Temperature-Dependent Förster Resonance Energy Transfer Studies on Rifampicin in an Anionic Sodium Dodecyl Sulfate Micelle

In this contribution, we report studies on the nature of binding of a potent antituberculosis drug, Rifampicin (RF) with a model drug delivery system, sodium dodecyl sulfate (SDS) micelle. Temperature dependent dynamic light scattering (DLS), conductometry, and circular dichroism (CD) spectroscopy have been employed to study the binding interaction of the drug with the micelle. The absorption spectrum of the drug RF in the visible region has been employed to study Förster resonance energy transfer (FRET) from another fluorescent drug Hoechst 33258 (H33258), bound to the micelle. Picosecond-resolved FRET studies at room temperature confirm the simultaneous binding of the two drugs to the micelle and the distance between the donor−acceptor pair is found to be 34 Å. The temperature dependent FRET study also confirms that the location and efficiency of drug binding to the micelle changes significantly at the elevated temperature. The energy transfer efficiency of the donor H33258, as measured from time-resolved studies, decreases significantly from 76% at 20 °C to 60% at 55 °C. This reveals detachment of some amount of the drug molecules from the micelles and increased donor−acceptor distance at elevated temperatures. The estimated donor−acceptor distance increases from a value of 33 Å at 20 °C to 37 Å at 55 °C. The picosecond resolved FRET studies on a synthesized DNA bound H33258 in RF solution have been performed to explore the interaction between the two. Our studies are expected to find relevance in the exploration of a potential vehicle for the vital drug rifampicin

Site- and sequence-selective ultrafast hydration of DNA

Water molecules in the DNA grooves are critical for maintaining structural integrity, conformational changes, and molecular recognition. Here we report studies of site- and sequence-specific hydration dynamics, using 2-aminopurine (Ap) as the intrinsic fluorescence probe and with femtosecond resolution. The dodecamer d[CGCA(Ap)ATTTGCG]2 was investigated, and we also examined the effect of a specific minor groove-binding drug, pentamidine, on hydration dynamics. Two time scales were observed: {approx}1 ps (bulk-like) and 10–12 ps (weakly bound type), consistent with layer hydration observed in proteins and DNA. However, for denatured DNA, the cosolvent condition of 40% formamide hydration is very different: it becomes that of bulk (in the presence of formamide). Well known electron transfer between Ap and nearby bases in stacked assemblies becomes inefficient in the single-stranded state. The rigidity of Ap in the single strands is significantly higher than that in bulk water and that attached to deoxyribose, suggesting a unique role for the dynamics of the phosphate-sugar-base in helix formation. The disparity in minor and major groove hydration is evident because of the site selection of Ap and in the time scale observed here (in the presence and absence of the drug), which is different by a factor of 2 from that observed in the minor groove–drug recognition

4D Scanning Ultrafast Electron Microscopy: Visualization of Materials Surface Dynamics

The continuous electron beam of conventional scanning electron microscopes (SEM) limits the temporal resolution required for the study of ultrafast dynamics of materials surfaces. Here, we report the development of scanning ultrafast electron microscopy (S-UEM) as a time-resolved method with resolutions in both space and time. The approach is demonstrated in the investigation of the dynamics of semiconducting and metallic materials visualized using secondary-electron images and backscattering electron diffraction patterns. For probing, the electron packet was photogenerated from the sharp field-emitter tip of the microscope with a very low number of electrons in order to suppress space–charge repulsion between electrons and reach the ultrashort temporal resolution, an improvement of orders of magnitude when compared to the traditional beam-blanking method. Moreover, the spatial resolution of SEM is maintained, thus enabling spatiotemporal visualization of surface dynamics following the initiation of change by femtosecond heating or excitation. We discuss capabilities and potential applications of S-UEM in materials and biological science

Photoselective excited state dynamics in ZnO–Au nanocomposites and their implications in photocatalysis and dye-sensitized solar cells

Improving the performance of photoactive solid-state devices begins with systematic studies of the metal–semiconductor nanocomposites (NCs) upon which such devices are based. Here, we report the photo-dependent excitonic mechanism and the charge migration kinetics in a colloidal ZnO–Au NC system. By using a picosecond-resolved Förster resonance energy transfer (FRET) technique, we have demonstrated that excited ZnO nanoparticles (NPs) resonantly transfer visible optical radiation to the Au NPs, and the quenching of defect-mediated visible emission depends solely on the excitation level of the semiconductor. The role of the gold layer in promoting photolytic charge transfer, the activity of which is dependent upon the degree of excitation, was probed using methylene blue (MB) reduction at the semiconductor interface. Incident photon-to-current efficiency measurements show improved charge injection from a sensitizing dye to a semiconductor electrode in the presence of gold in the visible region. Furthermore, the short-circuit current density and the energy conversion efficiency of the ZnO–Au NP based dye-sensitized solar cell (DSSC) are much higher than those of a DSSC comprised of only ZnO NP. Our results represent a new paradigm for understanding the mechanism of defect-state passivation and photolytic activity of the metal component in metal–semiconductor nanocomposite systems

Development of a Fiber Optic Sensor for Online Monitoring of Thin Coatings

 The thickness measurement of gas, liquid and solid layers is not only important for the basic research on nanoscience but equally valuable in contemporary applied biomedical research. Here, we have developed an optical spectroscopy based technique for the online monitoring of thin films (coatings). A low cost light emitting diode (LED) source combined with a fiber optic bundle and grating based spectrograph have been used to generate white light interferogram. We have monitored online change of refractive index of an air film (~4 μm thickness) with temperature following the change in the intensity profile of the interferogram. A thin film of water between two cover slips (thin glass plates) has also been monitored. We have proposed a schematic for further lowering the cost of the developed instrument for the online monitoring of the coating thickness (semitransparent liquid/gas/solid films) during manufacturing/processing. A brief theoretical analysis on the detection limit of the developed technique has also been discussed in the paper

Development Of Shape Memory Alloy Based Quarter Car Suspension System

It is well-known that suspension systems plays a major role in automotive technology. Most of the today’s vehicle applies a passive suspension systems consisting of a spring and damper. The design of automotive suspension have been a compromise between passenger comfort, suspension travel and road holding ability. This work aims in reducing the suspension travel alone by developing a quarter car model suspension for a passenger car to improve its performance by introducing shape memory alloy spring (Nitinol) instead of traditional spring. A two way shape memory alloy spring possesses two different stiffness in its two different phases (martensite and austenite). In this study, road profile is considered as a simple harmonic profile and vibration analysis of aminiature quarter car model suspension system has been carried out experimentally. Using theoretical method, the displacement of the sprung mass is also studied and discussed. The vibration analysis have been carried out for the suspension system at both phases of the spring and the results gives a significant improvement in reducing the displacement of sprung mass for various excitation frequencies

Femtosecond Studies of Protein-DNA Binding and Dynamics: Histone I

In this contribution, we report studies of the nature of binding interactions and dynamics of protein histone I (H1) with ligands in solution and as a complex with DNA, an important biological process for the higher-order structure in chromatin. With femtosecond time resolution, we examined the role of solvation by water, the micropolarity at the interface of the binding site(s) of H1, and the rigidity of the complex structure. We used two biologically common fluorescent probes: 2-(p-toluidino)naphthalene-6-sulfonate (TNS) and 5-(dimethylamino)naphthalene-1-sulfonyl chloride (DC). By noncovalently attaching TNS and covalently adducting DC to the binding sites we found that the solvation dynamics, which occur within 1 ps, for the probe at the protein surface and in bulk solution are comparable, indicating the significant contribution of bulk water shells. However, the local polarity changes significantly, reflecting the change in dielectric properties at the protein/water interface. The binding structure of the protein–DNA complex was examined by the local orientational motion of the probe. The covalently bound DC molecule, sandwiched between the protein and DNA, was found to be frozen, revealing the very rigid structure at the recognition site, while, for noncovalently bound TNS, the complexes displace the probe. The dynamical rigidity of the complex, and the role of solvation and interface polarity, elucidate the strong recognition mechanism between DNA and the protein by electrostatic interactions, which are important to the compactness and to chromatin condensation in the biological function