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- Creators: School of Molecular Sciences
- Member of: ASU Regents' Professors Open Access Works
X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory, exposing single inorganic core-shell nanoparticles to femtosecond hard-X-ray pulses. Each facetted nanoparticle consisted of a crystalline gold core and a differently shaped palladium shell. Scattered intensities were observed up to about 7 nm resolution. Analysis of the scattering patterns revealed the size distribution of the samples, which is consistent with that obtained from direct real-space imaging by electron microscopy. Scattering patterns resulting from single particles were selected and compiled into a dataset which can be valuable for algorithm developments in single particle scattering research.
Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a well-characterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 μm diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 Ångström were recorded. The diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development, the contents of which are described here.
Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/function questions remain regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe expression of Vpu in bacteria, its purification and characterization. We report the successful expression of PelB-Vpu in Escherichia coli using the leader peptide pectate lyase B (PelB) from Erwinia carotovora. The protein was detergent extractable and could be isolated in a very pure form. We demonstrate that the PelB signal peptide successfully targets Vpu to the cell membranes and inserts it as a type I membrane protein. PelB-Vpu was biophysically characterized by circular dichroism and dynamic light scattering experiments and was shown to be an excellent candidate for elucidating structural models.
Background:
Iron is an essential micronutrient for all organisms because it is a component of enzyme cofactors that catalyze redox reactions in fundamental metabolic processes. Even though iron is abundant on earth, it is often present in the insoluble ferric [Fe (III)] state, leaving many surface environments Fe-limited. The haploid green alga Chlamydomonas reinhardtii is used as a model organism for studying eukaryotic photosynthesis. This study explores structural and functional changes in PSI-LHCI supercomplexes under Fe deficiency as the eukaryotic photosynthetic apparatus adapts to Fe deficiency.
Results:
77K emission spectra and sucrose density gradient data show that PSI and LHCI subunits are affected under iron deficiency conditions. The visible circular dichroism (CD) spectra associated with strongly-coupled chlorophyll dimers increases in intensity. The change in CD signals of pigments originates from the modification of interactions between pigment molecules. Evidence from sucrose gradients and non-denaturing (green) gels indicates that PSI-LHCI levels were reduced after cells were grown for 72 h in Fe-deficient medium. Ultrafast fluorescence spectroscopy suggests that red-shifted pigments in the PSI-LHCI antenna were lost during Fe stress. Further, denaturing gel electrophoresis and immunoblot analysis reveals that levels of the PSI subunits PsaC and PsaD decreased, while PsaE was completely absent after Fe stress. The light harvesting complexes were also susceptible to iron deficiency, with Lhca1 and Lhca9 showing the most dramatic decreases. These changes in the number and composition of PSI-LHCI supercomplexes may be caused by reactive oxygen species, which increase under Fe deficiency conditions.
Conclusions:
Fe deficiency induces rapid reduction of the levels of photosynthetic pigments due to a decrease in chlorophyll synthesis. Chlorophyll is important not only as a light-harvesting pigment, but also has a structural role, particularly in the pigment-rich LHCI subunits. The reduced level of chlorophyll molecules inhibits the formation of large PSI-LHCI supercomplexes, further decreasing the photosynthetic efficiency.
The occurrence of nonliquid and liquid physical states of submicron atmospheric particulate matter (PM) downwind of an urban region in central Amazonia was investigated. Measurements were conducted during two intensive operating periods (IOP1 and IOP2) that took place during the wet and dry seasons of the GoAmazon2014/5 campaign. Air masses representing variable influences of background conditions, urban pollution, and regional- and continental-scale biomass burning passed over the research site. As the air masses varied, particle rebound fraction, an indicator of physical state, was measured in real time at ground level using an impactor apparatus. Micrographs collected by transmission electron microscopy confirmed that liquid particles adhered, while nonliquid particles rebounded. Relative humidity (RH) was scanned to collect rebound curves.
When the apparatus RH matched ambient RH, 95 % of the particles adhered as a campaign average. Secondary organic material, produced for the most part by the oxidation of volatile organic compounds emitted from the forest, produces liquid PM over this tropical forest. During periods of anthropogenic influence, by comparison, the rebound fraction dropped to as low as 60 % at 95 % RH. Analyses of the mass spectra of the atmospheric PM by positive-matrix factorization (PMF) and of concentrations of carbon monoxide, total particle number, and oxides of nitrogen were used to identify time periods affected by anthropogenic influences, including both urban pollution and biomass burning. The occurrence of nonliquid PM at high RH correlated with these indicators of anthropogenic influence. A linear model having as output the rebound fraction and as input the PMF factor loadings explained up to 70 % of the variance in the observed rebound fractions. Anthropogenic influences can contribute to the presence of nonliquid PM in the atmospheric particle population through the combined effects of molecular species that increase viscosity when internally mixed with background PM and increased concentrations of nonliquid anthropogenic particles in external mixtures of anthropogenic and biogenic PM.