Protein-Protein Affinity Determination by Quantitative FRET Quenching.

The molecular dissociation constant, Kd, is a well-established parameter to quantitate the affinity of protein-protein or other molecular interactions. Recently, we reported the theoretical basis and experimental procedure for Kd determination using a quantitative FRET method. Here we report a new development of Kd determination by measuring the reduction in donor fluorescence due to acceptor quenching in FRET. A new method of Kd determination was developed from the quantitative measurement of donor fluorescence quenching. The estimated Kd values of SUMO1-Ubc9 interaction based on this method are in good agreement with those determined by other technologies, including FRET acceptor emission. Thus, the acceptor-quenched approach can be used as a complement to the previously developed acceptor excitation method. The new methodology has more general applications regardless whether the acceptor is an excitable fluorophore or a quencher. Thus, these developments provide a complete methodology for protein or other molecule interaction affinity determinations in solution

Development of a Universal FRET Technology for Determining Biochemical and Pharmaceutical Parameters and Application in Deciphering the Interplay Between Influenza Viruses and SUMOylation Pathway

SUMOylation is one of the most important post-translational modifications, which plays pivotal roles in many physiological processes. SUMOylation is a multi-step enzymatic cascade, which involves multiple protein-protein interactions and regulates protein activity in many aspects. Misregulation of the SUMO pathway has been associated with many types of diseases, including viral infection, tumorigenesis, and neurodegenerative diseases. Thus, SUMOylation has great potential to be a target for the development of novel antiviral and anticancer agents. Förster Resonance Energy Transfer (FRET) is an energy transfer process that occurs between two interacting fluorophores with overlapping spectra, and is widely used to study protein-protein interactions. Previously, our group has developed quantitative FRET assay for determining protein interaction affinity and enzymatic kinetics. Here, my research goal is to further develop quantitative FRET (qFRET) technology into a robust and reliable method for determining biochemical parameters, and to investigate the interplay between SUMOylation and influenza replication. Specifically, I have proven our high-sensitive FRET technology can be used to determine protein interaction affinity even without protein purification, and utilizing this strategy I have for the first time measured the Kd between SUMO E3-PIAS1 with SUMO E2 or SUMO substrate-NS1 which elucidates the mechanism of how SUMO E3 regulates SUMOylation of different substrates. Then, I have developed a systematic method for determining mechanism of enzyme inhibition through our qFRET technology, and characterized the inhibition type and inhibition constant of our newly found SUMOylation inhibitor- STE. We have also identified Lys131 of influenza NS1 protein as the SUMOylated lysine residue which is important for virus replication through an in vitro FRET-based SUMOylation assay. And lastly, I have shown our SUMOylation inhibitor-STE exhibits great inhibition on influenza growth and can serve as a potential new anti-influenza drug. In summary, these findings prove our qFRET technology provides a powerful tool for determining biochemical parameters and dissects the role of SUMOylation in influenza viral life cycle

Cross-Scaling Approach for Water-Flow-Regulating Ecosystem Services: A Trial in Bochum, Germany

Water-flow-regulating ecosystem services (ESs) determine the regulation of hydrological flows on the ground’s surface. A lack of water-flow-regulating ESs would cause environmental problems such as heavy rainfall runoff and urban water logging, leading to floods affecting well-being, especially in dense urban areas. Research on water-flow-regulating ES supply–demand relationships in urban areas is urgently needed to better support the management of urban surface runoff. However, matching the supply–demand relationships of water-flow-regulating ESs remains challenging. In this contribution, a cross-scale approach linking the supply–demand assessment of water-flow-regulating ESs on a macroscale and the evaluation of the constructed urban environment on a microscale was developed. The approach was applied in the city of Bochum, Germany, as a trial of bridging the “science–practice gap”. Our findings show that the supply–demand budget of water-flow-regulating ESs in Bochum exhibits an urban–rural difference and is also partially influenced by land cover transformations such as vegetation degradation. In addition, further assessment of the constructed urban environment confirmed the result from the assessment of water-flow-regulating ESs based on the understanding of the urban hydrological cycle in Bochum. To account for the mismatch in the supply–demand budget, we classified the typical superior and inferior forms of urban water-flow-regulating ESs through field research on the same extreme areas to summarize the operable optimization, enhancement, and protection suggestions for urban construction decision makers. Finally, the cross-scale approach was approved as a possible way to bridge the “science–practice” gap for water-flow-regulating ES research in urban areas

Influence of Charge Hole Spacing on the Crack Propagation Behavior under the Effect of Empty-Hole Directional Blasting

In order to explore the reasonable spacing of charge holes in empty-hole directional blasting, based on the action mechanism of empty-hole directional blasting, the influence of charge hole spacing on crack propagation is studied, the calculation formula of charge hole spacing is deduced, and the blasting excavation process in Shijiazhuang South Ring Expressway Tunnel is simulated by finite element software. The results show that by setting empty holes on both sides of the charge holes in the peripheral blasting, the guiding cracks can be formed in the connecting direction between the empty hole and the charge hole, the propagation of cracks in other directions can be suppressed, and the spacing of charge holes can be enlarged. Reasonable charge hole spacing is very important to realize the effect of empty-hole directional blasting; the spacing is small, the rock will be overbroken, and the utilization ratio of explosives is reduced; large spacing, the guiding effect of empty holes is weak, and the effective directional cracks cannot be formed. The spacing between charge holes is closely related to the mechanical properties of rock, detonation parameters of explosive, and aperture of the charge hole and empty hole. According to the simulation results of crack propagation in different spacing, the blasting performance is the best when the charge hole spacing is 800–1000 mm, which is completely consistent with the theoretical calculation result of charge hole spacing 948 mm

Protein-Protein Affinity Determination by Quantitative FRET Quenching.

The molecular dissociation constant, Kd, is a well-established parameter to quantitate the affinity of protein-protein or other molecular interactions. Recently, we reported the theoretical basis and experimental procedure for Kd determination using a quantitative FRET method. Here we report a new development of Kd determination by measuring the reduction in donor fluorescence due to acceptor quenching in FRET. A new method of Kd determination was developed from the quantitative measurement of donor fluorescence quenching. The estimated Kd values of SUMO1-Ubc9 interaction based on this method are in good agreement with those determined by other technologies, including FRET acceptor emission. Thus, the acceptor-quenched approach can be used as a complement to the previously developed acceptor excitation method. The new methodology has more general applications regardless whether the acceptor is an excitable fluorophore or a quencher. Thus, these developments provide a complete methodology for protein or other molecule interaction affinity determinations in solution

Ultrafast Formation of Charge Transfer Trions at Molecular-Doped 2D MoS2 Interfaces

In this work, we examined trion dynamics at an organometallic molecule/monolayer transition metal dichalcogenides (TMDs) heterojunction, using transient electronic sum frequency generation spectroscopy. Theoretical analysis showed that the ground state bleach and stimulated emission signal from transient electronic sum frequency generation cancel each other due to the opposite sign – different from that of transient absorption – which makes this technique specifically sensitive to trion formation dynamics. Upon pumping at 2.4eV, we observed an ultrafast hole transfer, followed by the formation of charge-transfer trions. The existence of charge-transfer trion exists at molecular functionalized TMDs heterojunction monolayer opens the possibility of engineering the local electronic structures and dynamics of specific locations on TMDs and offers the potential for transferring unique electronic attributes of TMD to the molecular layers for molecular-based spintronics