942 research outputs found
Mechanical control of the directional stepping dynamics of the kinesin motor
Among the multiple steps constituting the kinesin's mechanochemical cycle,
one of the most interesting events is observed when kinesins move an 8-nm step
from one microtubule (MT)-binding site to another. The stepping motion that
occurs within a relatively short time scale (~100 microsec) is, however, beyond
the resolution of current experiments, therefore a basic understanding to the
real-time dynamics within the 8-nm step is still lacking. For instance, the
rate of power stroke (or conformational change), that leads to the
undocked-to-docked transition of neck-linker, is not known, and the existence
of a substep during the 8-nm step still remains a controversial issue in the
kinesin community. By using explicit structures of the kinesin dimer and the MT
consisting of 13 protofilaments (PFs), we study the stepping dynamics with
varying rates of power stroke (kp). We estimate that 1/kp <~ 20 microsec to
avoid a substep in an averaged time trace. For a slow power stroke with 1/kp>20
microsec, the averaged time trace shows a substep that implies the existence of
a transient intermediate, which is reminiscent of a recent single molecule
experiment at high resolution. We identify the intermediate as a conformation
in which the tethered head is trapped in the sideway binding site of the
neighboring PF. We also find a partial unfolding (cracking) of the binding
motifs occurring at the transition state ensemble along the pathways prior to
binding between the kinesin and MT.Comment: 26 pages, 10 figure
Universality and diversity of folding mechanics for three-helix bundle proteins
In this study we evaluate, at full atomic detail, the folding processes of
two small helical proteins, the B domain of protein A and the Villin headpiece.
Folding kinetics are studied by performing a large number of ab initio Monte
Carlo folding simulations using a single transferable all-atom potential. Using
these trajectories, we examine the relaxation behavior, secondary structure
formation, and transition-state ensembles (TSEs) of the two proteins and
compare our results with experimental data and previous computational studies.
To obtain a detailed structural information on the folding dynamics viewed as
an ensemble process, we perform a clustering analysis procedure based on graph
theory. Moreover, rigorous pfold analysis is used to obtain representative
samples of the TSEs and a good quantitative agreement between experimental and
simulated Fi-values is obtained for protein A. Fi-values for Villin are also
obtained and left as predictions to be tested by future experiments. Our
analysis shows that two-helix hairpin is a common partially stable structural
motif that gets formed prior to entering the TSE in the studied proteins. These
results together with our earlier study of Engrailed Homeodomain and recent
experimental studies provide a comprehensive, atomic-level picture of folding
mechanics of three-helix bundle proteins.Comment: PNAS, in press, revised versio
Microsecond folding dynamics of the F13W G29A mutant of the B domain of staphylococcal protein A by laser-induced temperature jump
The small size (58 residues) and simple structure of the B domain of staphylococcal protein A (BdpA) have led to this domain being a paradigm for theoretical studies of folding. Experimental studies of the folding of BdpA have been limited by the rapidity of its folding kinetics. We report the folding kinetics of a fluorescent mutant of BdpA (G29A F13W), named F13W*, using nanosecond laser-induced temperature jump experiments. Automation of the apparatus has permitted large data sets to be acquired that provide excellent signal-to-noise ratio over a wide range of experimental conditions. By measuring the temperature and denaturant dependence of equilibrium and kinetic data for F13W*, we show that thermodynamic modeling of multidimensional equilibrium and kinetic surfaces is a robust method that allows reliable extrapolation of rate constants to regions of the folding landscape not directly accessible experimentally. The results reveal that F13W* is the fastest-folding protein of its size studied to date, with a maximum folding rate constant at 0 M guanidinium chloride and 45°C of 249,000 (s-1). Assuming the single-exponential kinetics represent barrier-limited folding, these data limit the value for the preexponential factor for folding of this protein to at least ≈2 x 10(6) s(-1)
Markov dynamic models for long-timescale protein motion
Molecular dynamics (MD) simulation is a well-established method for studying protein motion at the atomic scale. However, it is computationally intensive and generates massive amounts of data. One way of addressing the dual challenges of computation efficiency and data analysis is to construct simplified models of long-timescale protein motion from MD simulation data. In this direction, we propose to use Markov models with hidden states, in which the Markovian states represent potentially overlapping probabilistic distributions over protein conformations. We also propose a principled criterion for evaluating the quality of a model by its ability to predict long-timescale protein motions. Our method was tested on 2D synthetic energy landscapes and two extensively studied peptides, alanine dipeptide and the villin headpiece subdomain (HP-35 NleNle). One interesting finding is that although a widely accepted model of alanine dipeptide contains six states, a simpler model with only three states is equally good for predicting long-timescale motions. We also used the constructed Markov models to estimate important kinetic and dynamic quantities for protein folding, in particular, mean first-passage time. The results are consistent with available experimental measurements
Advances in multispectral and hyperspectral imaging for archaeology and art conservation
Multispectral imaging has been applied to the field of art conservation and art history since the early 1990s. It is attractive as a noninvasive imaging technique because it is fast and hence capable of imaging large areas of an object giving both spatial and spectral information. This paper gives an overview of the different instrumental designs, image processing techniques and various applications of multispectral and hyperspectral imaging to art conservation, art history and archaeology. Recent advances in the development of remote and versatile multispectral and hyperspectral imaging as well as techniques in pigment identification will be presented. Future prospects including combination of spectral imaging with other noninvasive imaging and analytical techniques will be discussed
Construction and characterization of recombinant flaviviruses bearing insertions between E and NS1 genes
<p>Abstract</p> <p>Background</p> <p>The yellow fever virus, a member of the genus <it>Flavivirus</it>, is an arthropod-borne pathogen causing severe disease in humans. The attenuated yellow fever 17D virus strain has been used for human vaccination for 70 years and has several characteristics that are desirable for the development of new, live attenuated vaccines. We described here a methodology to construct a viable, and immunogenic recombinant yellow fever 17D virus expressing a green fluorescent protein variant (EGFP). This approach took into account the presence of functional motifs and amino acid sequence conservation flanking the E and NS1 intergenic region to duplicate and fuse them to the exogenous gene and thereby allow the correct processing of the viral polyprotein precursor.</p> <p>Results</p> <p>YF 17D EGFP recombinant virus was grew in Vero cells and reached a peak titer of approximately 6.45 ± 0.4 log10 PFU/mL at 96 hours post-infection. Immunoprecipitation and confocal laser scanning microscopy demonstrated the expression of the EGFP, which was retained in the endoplasmic reticulum and not secreted from infected cells. The association with the ER compartment did not interfere with YF assembly, since the recombinant virus was fully competent to replicate and exit the cell. This virus was genetically stable up to the tenth serial passage in Vero cells. The recombinant virus was capable to elicit a neutralizing antibody response to YF and antibodies to EGFP as evidenced by an ELISA test. The applicability of this cloning strategy to clone gene foreign sequences in other flavivirus genomes was demonstrated by the construction of a chimeric recombinant YF 17D/DEN4 virus.</p> <p>Conclusion</p> <p>This system is likely to be useful for a broader live attenuated YF 17D virus-based vaccine development for human diseases. Moreover, insertion of foreign genes into the flavivirus genome may also allow <it>in vivo </it>studies on flavivirus cell and tissue tropism as well as cellular processes related to flavivirus infection.</p
Capillary abnormalities in workers occupationally exposed to ionizing and nonionizing radiations
Cilj studije je bio metodom kapilaroskopije upozoriti na mogući utjecaj ionizacijskog i neionizacijskog zračenja na periferni krvotok profesionalno izloženih osoba. Kapilaroskopski smo pregledali 100 osoba profesionalno izloženih ionizacijskom zračenju, 110 osoba profesionalno izloženih neionizacijskom zračenju i 80 kontrolnih, neizloženih osoba. U obje test-skupine oštećenja mikrocirkulacije su bila statistički značajno učestalija nego u kontrolnoj skupini.The aim of the study was to determine a possible effect of ionizing and nonionizing radiations on peripheral blood flow in occupationally exposed persons by means of capillaroscopic analysis. Altogether 290 subjects were examined. Of these 100 were occupationally exposed to ionizing radiation, 110 were occupationally exposed to nonionizing radiation and 80 control subjects never worked with radiation sources. Statistical analysis showed that microvascular abnormalities occurred significantly more frequently among occupationally exposed persons than in the control group
Plasmonically Enhanced Reflectance of Heat Radiation from Low-Bandgap Semiconductor Microinclusions
Increased reflectance from the inclusion of highly scattering particles at
low volume fractions in an insulating dielectric offers a promising way to
reduce radiative thermal losses at high temperatures. Here, we investigate
plasmonic resonance driven enhanced scattering from microinclusions of
low-bandgap semiconductors (InP, Si, Ge, PbS, InAs and Te) in an insulating
composite to tailor its infrared reflectance for minimizing thermal losses from
radiative transfer. To this end, we compute the spectral properties of the
microcomposites using Monte Carlo modeling and compare them with results from
Fresnel equations. The role of particle size-dependent Mie scattering and
absorption efficiencies, and, scattering anisotropy are studied to identify the
optimal microinclusion size and material parameters for maximizing the
reflectance of the thermal radiation. For composites with Si and Ge
microinclusions we obtain reflectance efficiencies of 57 - 65% for the incident
blackbody radiation from sources at temperatures in the range 400 - 1600
{\deg}C. Furthermore, we observe a broadbanding of the reflectance spectra from
the plasmonic resonances due to charge carriers generated from defect states
within the semiconductor bandgap. Our results thus open up the possibility of
developing efficient high-temperature thermal insulators through use of the
low-bandgap semiconductor microinclusions in insulating dielectrics.Comment: Main article (8 Figures and 2 Tables) + Supporting Information (8
Figures
Two-Dimensional Infrared Spectroscopy of Antiparallel β-Sheet Secondary Structure
We investigate the sensitivity of femtosecond Fourier transform two-dimensional infrared spectroscopy to protein secondary structure with a study of antiparallel β-sheets. The results show that 2D IR spectroscopy is more sensitive to structural differences between proteins than traditional infrared spectroscopy, providing an observable that allows comparison to quantitative models of protein vibrational spectroscopy. 2D IR correlation spectra of the amide I region of poly-L-lysine, concanavalin A, ribonuclease A, and lysozyme show cross-peaks between the IR-active transitions that are characteristic of amide I couplings for polypeptides in antiparallel hydrogen-bonding registry. For poly-L-lysine, the 2D IR spectrum contains the eight-peak structure expected for two dominant vibrations of an extended, ordered antiparallel β-sheet. In the proteins with antiparallel β-sheets, interference effects between the diagonal and cross-peaks arising from the sheets, combined with diagonally elongated resonances from additional amide transitions, lead to a characteristic “Z”-shaped pattern for the amide I region in the 2D IR spectrum. We discuss in detail how the number of strands in the sheet, the local configurational disorder in the sheet, the delocalization of the vibrational excitation, and the angle between transition dipole moments affect the position, splitting, amplitude, and line shape of the cross-peaks and diagonal peaks.
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