Ligand-dependent folding and unfolding dynamics and free energy landscapes of acylphosphatase.

Acylphosphatase (AcP) is an enzyme which catalyses the hydrolysis of acylphosphate. The binding with the phosphate ion (Pi) assumes significance in preserving both the stability and enzymatic activity of AcP. While previous studies using single molecule force spectroscopy explored the mechanical properties of AcP, the influence of Pi on its folding and unfolding dynamic behaviors remains unexplored. In this work, using stable magnetic tweezers, we measured and compared the force-dependent folding and unfolding rates of AcP in the Tris buffer and phosphate buffer within a force range from 2 pN to 40 pN. We found that Pi exerts no discernible effect on the folding dynamics but consistently decreases the force-dependent unfolding rate of AcP by a constant ratio across the entire force spectrum. The free energy landscapes of AcP in the absence and presence of Pi are constructed. Our results reveal that Pi selectively binds to the native state of AcP, stabilizing it and suggesting the general properties of specific ligand-receptor interactions.

Study on stress and deformation characteristics of existing-new two-stage cantilever retaining wall.

A two-stage cantilever retaining wall is composed of two single-stage cantilever retaining walls, which are stacked up and down. The structure not only has the advantages of a single-stage retaining wall, but also compensates for the shortcomings of the height limit of the single-stage retaining wall; therefore, it has been gradually applied in projects. Based on the actual project of Zhongwei-Lanzhou Passenger Dedicated Line into Lanzhou Hub, this paper studies the influence of the construction of new cantilever retaining wall and the filling of subgrade on the deformation and earth pressure of the new cantilever wall and the existing cantilever wall by means of field test and numerical simulation. The results show that with an increase in the filling height after the new cantilever wall (upper wall), the horizontal displacement of the top of the upper and lower walls increased nonlinearly. The displacement direction of the upper wall was the filling direction, and that of the lower wall was the deviation from the filling direction. The higher the filling height, the greater is the displacement. With an increase in the filling height, the earth pressure behind the upper wall increases gradually along the wall height and decreases slightly to the bottom of the wall, which is approximately a linear distribution. The earth pressure behind the existing cantilever wall first increases along the wall height and gradually decreases after reaching a certain depth, but the earth pressure of the lower wall does not increase significantly with an increase in the filling height behind the upper wall. The slope failure mode is the overall sliding failure of the retaining wall together with the fill soil. The sliding surface passed through the lower edge of the lower wall heel and was similar to an arc shape. The stability of the two-stage cantilever retaining wall was better than that of a single-stage retaining wall. Finally, a calculation method for the overall stability and earth pressure of the existing two-stage cantilever retaining wall was proposed.

CBL1/9-CIPK23-NRAMP1 axis regulates manganese toxicity.

Manganese (Mn) is an essential micronutrient in plants. However, excessive Mn absorption in acidic soils can cause Mn toxicity, which adversely affects plant growth and crop yields. At present, acidic soils cover c. 30% of the Earth's surface. However, the mechanism underpinning Mn uptake remains largely unknown. We identified cbl1/9 and cipk23 mutants exhibiting high-Mn-sensitive phenotype through the reverse genetics method. Furthermore, we identified the CIPK23 phosphorylated NRAMP1 through a variety of protein interaction techniques and protein kinase assays. Here, we demonstrated that two calcineurin B-like proteins, CBL1/9, and their interacting kinase CIPK23 positively regulated the tolerance of Mn toxicity in Arabidopsis. The cbl1 cbl9 double mutant and cipk23 mutants exhibited high-Mn-sensitive phenotypes, which manifested as decreased primary root length, biomass, and chlorophyll concentration, and higher accumulation of Mn. In addition, CIPK23 interacted with and phosphorylated the Mn transporter NRAMP1 primarily at Ser20/22 in vitro and in vivo, and thereby induced clathrin-mediated endocytosis of NRAMP1 to reduce its distribution on the plasma membrane and enhance plant tolerance to Mn toxicity. In summary, we found that the CBL1/9-CIPK23-NRAMP1 module regulates the tolerance to high-Mn toxicity and provide insight into a mechanism of the tolerance of plants to Mn toxicity.

Free-energy landscape of two-state protein acylphosphatase with large contact order revealed by force-dependent folding and unfolding dynamics.

Acylphosphatase (AcP) is a small protein with 98 amino acid residues that catalyzes the hydrolysis of carboxyl-phosphate bonds. AcP is a typical two-state protein with slow folding rate due to its relatively large contact order in the native structure. The mechanical properties and unfolding behavior of AcP has been studied by atomic force microscope. Here using stable magnetic tweezers, we measured the force-dependent folding rates within a force range 1-3 pN, and unfolding rates 15-40 pN. The obtained unfolding rates show different force sensitivities at forces below and above ∼27 pN, which determines a free-energy landscape with two energy barriers. Our results indicate that the free-energy landscape of small globule proteins have general Bactrian camel shape, and large contact order of the native state produces a high barrier dominate at low forces.

Physiological and proteomic responses of Chlamydomonas reinhardtii to arsenate and lead mixtures.

Arsenic (As) and lead (Pb) are frequently emitted from various sources into environment, but microbial responses to their combined toxicity have not been systematically investigated. In this study, Chlamydomonas reinhardtii was exposed to two levels of arsenate (As (V), 50, 500 µg/L), Pb (II) (500, 5000 µg/L) and their mixture (50 µg/L As (V) + 500 µg/L Pb (II); 500 µg/L As (V) + 5000 µg/L Pb (II)). The growth of C. reinhardtii was inhibited more remarkably by As (V) than by Pb (II). The As stress was alleviated by Pb in the 50 µg/L As (V) + 500 µg/L Pb (II) treatment, but was enhanced upon the 500 µg/L As (V) + 5000 µg/L Pb (II) exposure, with more pronounced changes in a number of physiological parameters of the algal cells. Proteomic results showed that 71 differently expressed proteins (DEPs) in the treatment of 50 µg/L As (V) + 500 µg/L Pb (II), and 167 DEPs were identified in that of 500 µg/L As (V) + 5000 µg/L Pb (II). These proteins were involved in energy metabolism, photosynthetic carbon fixation, reactive oxygen scavenging and defense, and amino acid synthesis. Taken together, these physiological and proteomic data demonstrated that C. reinhardtii could resist the As (V) and Pb (II) combined treatments through extracellular complexation and intracellular pathways.

Contributions of polysaccharides to arsenate resistance in Chlamydomonas reinhardtii.

Polysaccharides supply energy for various metabolic processes in cells. However, their roles in the arsenate (As(V)) resistance in microalgae remain largely unknown. Here, we explored the synthesis and transformation of polysaccharides in Chlamydomonas reinhardtii upon various levels of As(V) stress, using a number of physiological indexes along with transmission electron microscopic (TEM) and proteomic analyses. When exposed to low concentration of As(V) (0-20 µg/L), C. reinhardtii accumulated starch and produced more extracellular polysaccharides. At 50 µg/L As(V) treatment, starch accumulation gradually shifted to polysaccharides decomposition in the algal cells. Under higher As(V) concentration (500 µg/L), significantly more proteins in fatty acid metabolic pathway were differentially expressed, indicating that cells redirected carbon flux and transformed lipids into polysaccharides. The findings of this study demonstrate that polysaccharides may be critically involved in the As(V) resistance of C. reinhardtii.

Two energy barriers and a transient intermediate state determine the unfolding and folding dynamics of cold shock protein.

Cold shock protein (Csp) is a typical two-state folding model protein which has been widely studied by biochemistry and single molecule techniques. Recently two-state property of Csp was confirmed by atomic force microscopy (AFM) through direct pulling measurement, while several long-lifetime intermediate states were found by force-clamp AFM. We systematically studied force-dependent folding and unfolding dynamics of Csp using magnetic tweezers with intrinsic constant force capability. Here we report that Csp mostly folds and unfolds with a single step over force range from 5 pN to 50 pN, and the unfolding rates show different force sensitivities at forces below and above ~8 pN, which determines a free energy landscape with two barriers and a transient intermediate state between them along one transition pathway. Our results provide a new insight on protein folding mechanism of two-state proteins.

[Characteristics of delta18O in precipitation and moisture transports over the arid region in Northwest China].

Based on the observed data of stable isotopes in precipitation and meteorological statistics at Urümqi (1986-2003), Zhangye (1986-2003), and Hotan (1988-1992) from IAEA/WMO, the precipitation equation of arid region in Northwest China was calculated through the regression analysis, and the characteristics of stable isotopic in precipitation, as well as the correlation between stable isotopic and meteorological records, were analyzed. The water vapor source of the three sites was tracked by the HYSPLIT4. 9 model, and water vapour transmission regime of arid region in Northwest China was established, while the record of delta18O in cryosphere was discussed. Results showed that the precipitation equation of deltaD = 7.24 delta18O + 1.96 per thousand indicates intense evaporation; delta18O values at Urümqi, Zhangye and Hotan fluctuates at -20.58 per thousand- -5.4 per thousand, -18.58 per thousand- -2.46 per thousand and -20.33 per thousand- -0.01 per thousand, respectively. The delta18O value is low in winter and high in summer, but the values of d-excess is on the contrary; The spatial distribution shows that delta18O value at Zhangye is highest, followed by Hotan, and the lowest is at Urümqi; The highest of d-excess value is at Urümqi, followed by Hotan, the lowest is at Zhangye; The temperature effect of delta18O in precipitation is significant with the correlation coefficients of 0.85, 0.81 and 0.86, respectively, and the rainfall effect does not exist; The modeled trajectory of vapor source showed there are two main paths all the year, which are from the Atlantic Ocean via Westerlies and the Arctic region, respectively, influence by different vapor sources, the seasonality of delta18O values lower in winter, higher in summer; In addition, according to the cryospheric environment, the correlation between delta18O in snow pack or ice core and the corresponding meteorological station record was significant, which reflects the variation of temperature efficiently.