Interaction of Chandipura Virus N and P Proteins: Identification of Two Mutually Exclusive Domains of N Involved in Interaction with P

The nucleocapsid protein (N) and the phosphoprotein (P) of nonsegmented negative-strand (NNS) RNA viruses interact with each other to accomplish two crucial events necessary for the viral replication cycle. First, the P protein binds to the aggregation prone nascent N molecules maintaining them in a soluble monomeric (N0) form (N0-P complex). It is this form that is competent for specific encapsidation of the viral genome. Second, the P protein binds to oligomeric N in the nucleoprotein complex (N-RNA-P complex), and thereby facilitates the recruitment of the viral polymerase (L) onto its template. All previous attempts to study these complexes relied on co-expression of the two proteins in diverse systems. In this study, we have characterised these different modes of N-P interaction in detail and for the first time have been able to reconstitute these complexes individually in vitro in the chandipura virus (CHPV), a human pathogenic NNS RNA virus. Using a battery of truncated mutants of the N protein, we have been able to identify two mutually exclusive domains of N involved in differential interaction with the P protein. An unique N-terminal binding site, comprising of amino acids (aa) 1–180 form the N0-P interacting region, whereas, C-terminal residues spanning aa 320–390 is instrumental in N-RNA-P interactions. Significantly, the ex-vivo data also supports these observations. Based on these results, we suggest that the P protein acts as N-specific chaperone and thereby partially masking the N-N self-association region, which leads to the specific recognition of viral genome RNA by N0

Understanding the spatio-temporal evolution of fractures in pillow basalt

We investigated the origin and spatio-temporal evolution of cooling fractures in pillow basalt which undergo thermal contraction after their eruption in an aqueous environment. Through a computer-based simulation using Fourier transformation, the thermo elastic stress displacement profiles within individual pillow units are determined. The scaled model (pillow diameter - 1 meter) generated radial, linear fractures perpendicular to pillow margin and irregular discrete flaws in the pillow interior like the ones observed in natural examples. Radial linear fractures of 3–5 centimetre in length have been measured in pillows of average one-metre diameter from the Maradihalli region, in the Chitradurga Schist Belt, India. An estimated time of 94–118 minutes was required to get radial fractures of similar length in the simulation. Our model efficiently replicated the generation and distribution of thermal fractures and allowed an estimation of cooling time for the peripheral glassy zone but has limitations in deciphering the formation of fracture networks in progressively crystalline inner zone of pillows

Understanding the spatio-temporal evolution of fractures in pillow basalt

We investigated the origin and spatio-temporal evolution of cooling fractures in pillow basalt which undergo thermal contraction after their eruption in an aqueous environment. Through a computer-based simulation using Fourier transformation, the thermo elastic stress displacement profiles within individual pillow units are determined. The scaled model (pillow diameter - 1 meter) generated radial, linear fractures perpendicular to pillow margin and irregular discrete flaws in the pillow interior like the ones observed in natural examples. Radial linear fractures of 3-5 centimetre in length have been measured in pillows of average one-metre diameter from the Maradihalli region, in the Chitradurga Schist Belt, India. An estimated time of 94-118 minutes was required to get radial fractures of similar length in the simulation. Our model efficiently replicated the generation and distribution of thermal fractures and allowed an estimation of cooling time for the peripheral glassy zone but has limitations in deciphering the formation of fracture networks in progressively crystalline inner zone of pillows

LoRUS: A Mobile Crowdsourcing System for Efficiently Retrieving the Top-k Relevant Users in a Spatial Window

The prevalence of mobile devices and applications strongly motivate mobile crowdsourcing for facilitating location-dependent services. We propose LoRUS, a Location-based Relevant User determination System for efficiently retrieving the top-k relevant mobile users in a given spatial window

Probing deuterium isotope effect on structure and solvation dynamics of human serum albumin

The deuterium isotopic effect on the structure and solvation dynamics of the protein, human serum albumin (HSA), has been studied by using circular dichroism (CD), femtosecond up-conversion, FRET, and single-molecule spectroscopy. The CD spectra suggest that D2O affects the structure of HSA, leading to a 20% decrease in the helical structure. The FRET study indicates that the distance of C153 from the lone tryptophan residue of HSA is quite similar ( ≈21 Å) in H2O and D2O, and hence, the location of the probe in the protein remains the same in the two solvents. The single-molecule study suggests that coumarin 153 (C153) binds almost exclusively (>96%) to one site of HSA. Solvation dynamics of C153 in HSA is found to be markedly retarded in D2O compared with H2O. In H2O, the solvation of C153 bound to HSA is found to be biexponential with one component of 7 ps (30%) and a long component of 350 ps (70%). In D2O, we detected a short component of 4 ps (41%) and a long component of 950 ps (59%). Thus, the ultraslow component of the solvation dynamics of C153 bound to HSA in D2O (950 ps) is 2.5-fold slower than that in H2O (350 ps). The marked deuterium isotope effect has been ascribed to water molecules confined in the protein environment and to a lesser extent to the structural modification of protein by D2O

Excited state proton transfer in ionic liquid mixed micelles

Excited state proton transfer (ESPT) of pyranine (8-hydroxypyranine-1,3,6-trisulfonate, HPTS) in room temperature ionic liquid (RTIL) mixed micelles is studied by femtosecond up-conversion. The mixed micelle consists of a triblock copolymer, (PEO)20-(PPO)70-(PEO)20 (Pluronic P123), and one of the two RTILs, 1-pentyl-3-methyl-imidazolium bromide ([pmim][Br]) and 1-pentyl-3-methyl-imidazolium tetra-fluoroborate ([pmim][BF4]). The size and structure of the mixed micelle vary with the relative amount of the RTIL. For [pmim][Br], the hydrodynamic diameter of the mixed micelle is 26 nm in 0.3 M RTIL and 3500 nm in 3.0 M RTIL. The time constant of initial proton transfer (τPT) in P123 micelle (65 ps) is 10 times slower than that (5 ps) in water, while the time constants of recombination (trec) and dissociation (τdiss) are 2-3 times slower in P123 micelle. On addition of the RTIL, the rate of ESPT is markedly modified. In 0.3 M RTIL-P123 mixed micelle, τPT is shorter than that in P123 micelle. In the mixed micelle, τPT increases with an increase in the concentration of the RTIL (230 ps in 3 M [pmim][Br] and 55 ps in 0.9 M [pmim][BF4]). This is attributed to large scale penetration of the P123 micelle by RTIL replacing water molecules. The time constants of proton transfer (τPT, τrec, and τdiss) are faster than the slowest component (200-500 ps) of solvation dynamics. It seems that the ultrafast component of solvation (<0.3 ps and <5 ps) is enough for inducing proton transfer. The time constant of the proton transfer (τPT) in [pmim][BF4]-P123 mixed micelle is longer (~20%) than that in [pmim][Br]-P123 mixed micelle for the same concentration of RTIL. The counterion dependence of ESPT is attributed to the difference in the structure and greater hydrophobicity of the [pmim][BF4]

Femtosecond study of ultrafast fluorescence resonance energy transfer in a catanionic vesicle

Ultrafast fluorescence resonance energy transfer (FRET) in a catanionic [sodium dodecyl sulfate (SDS)-dodecyltrimethyl ammonium bromide (DTAB)] vesicle is studied by femtosecond up-conversion. The vesicles (diameter ~400 nm for SDS-rich and ~250 nm for DTAB-rich vesicles) are much larger than the SDS and DTAB micelles (diameter ~4 nm). In both micelle and vesicles, FRET occurs in multiple time scales and the time scales of FRET correspond to a donor-acceptor distance varying between 12 and 36 Å

Effect of room temperature ionic liquids on femtosecond solvation dynamics in a triblock copolymer (P123) gel

1047-1055Effect of two room temperature ionic liquids (RTIL, [pmim][Br] and [pmim][BF4]) on the solvation dynamics and anisotropy decay of coumarin 480 (C480) in P123 gel has been studied using femtosecond up-conversion. In P123 gel, C480 displays a significant excitation wavelength dependence of emission maximum (red edge excitation shift, REES of 18 nm) and solvation dynamics, which suggest heterogeneous structure of the P123 gel. Dynamics of different regions of the P123 gel-RTIL is probed by the variation of excitation wavelength (ex). For ex = 435 nm, the polar peripheral region of the P123-RTIL gel is preferentially excited with a very small contribution of the non-polar region. At ex = 375 nm, contribution of the non-polar region is very large. The solvation dynamics in both the core and corona regions of P123 gel-RTIL is faster than that in P123 gel. The anisotropy decay of C480 in the P123 gel-RTIL phase is about two times faster as compared to P123 gel. The results are compared with a recent fluorescence correlation spectroscopic study on diffusion in RTIL-P123 gel. </span

Binding of organic dyes with human serum albumin: a single-molecule study

Kinetics of binding of dyes at different sites of human serum albumin (HSA) has been studied by single-molecule spectroscopy. The protein was immobilized on a glass surface. To probe different binding sites (hydrophobic and hydrophilic) two dyes, coumarin 153 (C153, neutral) and rhodamine 6G (R6G, cationic) were chosen. For both the dyes, a major (ca. 96-98%) and minor (ca. 3%) binding site were detected. Rate constants of association and dissociation were simultaneously determined from directly measuring fluctuations in fluorescence intensity (τ<SUB>off</SUB> and τ<SUB>on</SUB>) and from this the equilibrium (binding) constants were calculated. Fluorescence lifetimes at individual sites were obtained from burst-integrated lifetime analysis. Distributions of lifetime histograms for both the probes (C153 and R6G) exhibit two maxima, which indicates the presence of two binding domains in the protein. Unfolding of the protein has been studied by adding guanidinium hydrochloride (GdnHCl) to the solution. It is observed that addition of GdnHCl affects the dissociation and association kinetics and hence, binding equilibrium of the association of C153. However, the effect of binding of R6G is not affected much. It is proposed that GdnHCl affects the hydrophobic binding sites more than the hydrophilic site

Ultrafast and ultraslow proton transfer of pyranine in an ionic liquid microemulsion

Effect of a room temperature ionic liquid (RTIL) and water on the ultrafast excited state proton transfer (ESPT) of pyranine (8-hydroxypyrene-1,3,6-trisulfonate, HPTS) inside a microemulsion is studied by femtosecond up-conversion. The microemulsion consists of the surfactant, triton X-100 (TX-100) in benzene (bz) and contains the RTIL, 1-pentyl-3-methyl-imidazolium tetrafluoroborate ([pmim] [BF4]) as the polar phase. In the absence of water, HPTS undergoes ultrafast ESPT inside the RTIL microemulsion (RTIL/TX-100/bz) and the deprotonated form (RO-) exhibits three rise components of 0.3, 14, and 375 ps. It is proposed that in the RTIL microemulsion, HPTS binds to the TX-100 at the interface region and participates in ultrafast ESPT to the oxygen atoms of TX-100. On addition of water an additional slow rise of 2150 ps is observed. Similar long rise component is also observed in water/TX-100/benzene reverse micelle (in the absence of [pmim] [BF4]). It is suggested that the added water molecules preferentially concentrate (trapped) around the palisade layer of the RTIL microemulsion. The trapped water molecules remain far from the HPTS both in the presence and absence of ionic liquid and gives rise to the slow component (2150 ps) of ESPT. Replacement of H2O by D2O causes an increase in the time constant of the ultraslow rise to 2350 ps