Evolution of a plant-specific copper chaperone family for chloroplast copper homeostasis

Metallochaperones traffic copper (Cu(+)) from its point of entry at the plasma membrane to its destination. In plants, one destination is the chloroplast, which houses plastocyanin, a Cu-dependent electron transfer protein involved in photosynthesis. We present a previously unidentified Cu(+) chaperone that evolved early in the plant lineage by an alternative-splicing event of the pre-mRNA encoding the chloroplast P-type ATPase in Arabidopsis 1 (PAA1). In several land plants, recent duplication events created a separate chaperone-encoding gene coincident with loss of alternative splicing. The plant-specific Cu(+) chaperone delivers Cu(+) with specificity for PAA1, which is flipped in the envelope relative to prototypical bacterial ATPases, compatible with a role in Cu(+) import into the stroma and consistent with the canonical catalytic mechanism of these enzymes. The ubiquity of the chaperone suggests conservation of this Cu(+)-delivery mechanism and provides a unique snapshot into the evolution of a Cu(+) distribution pathway. We also provide evidence for an interaction between PAA2, the Cu(+)-ATPase in thylakoids, and the Cu(+)-chaperone for Cu/Zn superoxide dismutase (CCS), uncovering a Cu(+) network that has evolved to fine-tune Cu(+) distribution

Evolution of a plant-specific copper chaperone family for chloroplast copper homeostasis

Metallochaperones traffic copper (Cu(+)) from its point of entry at the plasma membrane to its destination. In plants, one destination is the chloroplast, which houses plastocyanin, a Cu-dependent electron transfer protein involved in photosynthesis. We present a previously unidentified Cu(+) chaperone that evolved early in the plant lineage by an alternative-splicing event of the pre-mRNA encoding the chloroplast P-type ATPase in Arabidopsis 1 (PAA1). In several land plants, recent duplication events created a separate chaperone-encoding gene coincident with loss of alternative splicing. The plant-specific Cu(+) chaperone delivers Cu(+) with specificity for PAA1, which is flipped in the envelope relative to prototypical bacterial ATPases, compatible with a role in Cu(+) import into the stroma and consistent with the canonical catalytic mechanism of these enzymes. The ubiquity of the chaperone suggests conservation of this Cu(+)-delivery mechanism and provides a unique snapshot into the evolution of a Cu(+) distribution pathway. We also provide evidence for an interaction between PAA2, the Cu(+)-ATPase in thylakoids, and the Cu(+)-chaperone for Cu/Zn superoxide dismutase (CCS), uncovering a Cu(+) network that has evolved to fine-tune Cu(+) distribution

A micro-patterned silicon chip as sample holder for macromolecular crystallography experiments with minimal background scattering

At low emittance synchrotron sources it has become possible to perform structure determinations from the measurement of multiple microcrystals which were previously considered too small for diffraction experiments. Conventional mounting techniques do not fulfill the requirements of these new experiments. They significantly contribute to background scattering and it is difficult to locate the crystals, making them incompatible with automated serial crystallography. We have developed a micro-fabricated sample holder from single crystalline silicon with micropores, which carries up to thousands of crystals and significantly reduces the background scattering level. For loading, the suspended microcrystals are pipetted onto the chip and excess mother liquor is subsequently soaked off through the micropores. Crystals larger than the pore size are retained and arrange themselves according to the micropore pattern. Using our chip we were able to collect 1.5 Å high resolution diffraction data from protein microcrystals with sizes of 4 micrometers and smaller

A micro-patterned silicon chip as sample holder for macromolecular crystallography experiments with minimal background scattering

At low emittance synchrotron sources it has become possible to perform structure determinations from the measurement of multiple microcrystals which were previously considered too small for diffraction experiments. Conventional mounting techniques do not fulfill the requirements of these new experiments. They significantly contribute to background scattering and it is difficult to locate the crystals, making them incompatible with automated serial crystallography. We have developed a micro-fabricated sample holder from single crystalline silicon with micropores, which carries up to thousands of crystals and significantly reduces the background scattering level. For loading, the suspended microcrystals are pipetted onto the chip and excess mother liquor is subsequently soaked off through the micropores. Crystals larger than the pore size are retained and arrange themselves according to the micropore pattern. Using our chip we were able to collect 1.5 Å high resolution diffraction data from protein microcrystals with sizes of 4 micrometers and smaller

Time-resolved pump and probe x-ray absorption fine structure spectroscopy at beamline P11 at PETRA III

We report about the development and implementation of a new setup for time-resolved X-ray absorption fine structure spectroscopy at beamline P11 utilizing the outstanding source properties of the low-emittance PETRA III synchrotron storage ring in Hamburg. Using a high intensity micrometer-sized X-ray beam in combination with two positional feedback systems, measurements were performed on the transition metal complex fac-Tris[2-phenylpyridinato-C2,N]iridium(III) also referred to as fac-Ir(ppy)3. This compound is a representative of the phosphorescent iridium(III) complexes, which play an important role in organic light emitting diode (OLED) technology. The experiment could directly prove the anticipated photoinduced charge transfer reaction. Our results further reveal that the temporal resolution of the experiment is limited by the PETRA III X-ray bunch length of ∼103 ps full width at half maximum (FWHM)