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In order to investigate the effects of Manganese (Mn) toxicity stress on the growth and gene expression at the seedling stage of soybean, soybean seedlings were treated with normal Mn concentration (5 μmol/L MnSO4) and excess Mn concentration (100 μmol/L MnSO4) by the method of hydroponic culture in this study. When soybean was subjected to Mn toxicity stress, excessive Mn could affect seedling growth, root development, the number of Mn oxide spots in leaves, and the Mn accumulation content in different parts of soybean. With the increase of exogenous Mn concentration and the prolongation of culture time, the shoot and root biomasses of soybean decreased significantly, but the plant height of soybean had no obvious effects. The total root length, root surface area and root volume of soybean decreased significantly, but the taproot length, taproot tip length and root diameter did not change significantly. The number of Mn spots in the primary leaves (first leaf) was significantly more than that in the old leaves (second leaf) and the youngest leaves (fourth leaf). The Mn concentration in leaves was significantly higher than that in roots, and the Mn concentration in the old leaves was significantly higher than that in youngest leaves with the method of inductively coupled plasma atomic emission spectrometry (ICP-AES). Moreover, the results in the present study suggested that the 10 selected genes were significantly up-regulated or down-regulated by Mn toxicity in the old and young leaves by quantitative real-time PCR (qRT-PCR) analysis. This indicates that these genes might be important in the process of regulation in old and young leaves of the physiological responses and ion transporting to Mn toxicity stress. 相似文献
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The folding pathway of the third domain of PDZ from the synaptic protein PSD-95 was characterized using kinetic and equilibrium methods by monitoring the fluorescence signal from a Trp residue that is incorporated at a near-surface position. Kinetic folding of this domain showed multiple exponential phases, whereas unfolding showed a single exponential phase. The slow kinetic phases were attributed to isomerization of proline residues, since there are five proline residues in this domain. We found that the logarithms of the rate constants for the fast phase of folding and unfolding are linearly dependent on the concentrations of denaturant. The unfolding free energy derived from these rate constants at zero denaturant was close to the value measured using the equilibrium method, suggesting the absence of detectable sub-millisecond folding intermediates. However, native-state hydrogen exchange experiments detected a partially unfolded intermediate under native conditions. It was further confirmed by a protein engineering study. These data suggest that a hidden intermediate exists after the rate-limiting step in the folding of the third domain of PDZ. 相似文献
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The focal adhesion target (FAT) domain of focal adhesion kinase has a four-helix bundle structure. Based on a hydrogen exchange-constrained computer simulation study and some indirect experimental results, it has been suggested that a partially unfolded state of the FAT domain with the N-terminal helix unfolded plays an important role in its biological function. Here, using a native-state hydrogen exchange method, we directly detected an intermediate with the N-terminal helix unfolded in a mutant (Y925E) of the FAT domain. In addition, kinetic folding studies on the FAT domain suggest that this intermediate exists on the native side of the rate-limiting transition state for folding. These results provide more direct evidence of the existence of the proposed intermediate and help to understand the folding mechanism of small single domain proteins. 相似文献
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Structures of intermediates and transition states in protein folding are usually characterized by amide hydrogen exchange and protein engineering methods and interpreted on the basis of the assumption that they have native-like conformations. We were able to stabilize and determine the high-resolution structure of a partially unfolded intermediate that exists after the rate-limiting step of a four-helix bundle protein, Rd-apocyt b(562), by multidimensional NMR methods. The intermediate has partial native-like secondary structure and backbone topology, consistent with our earlier native state hydrogen exchange results. However, non-native hydrophobic interactions exist throughout the structure. These and other results in the literature suggest that non-native hydrophobic interactions may occur generally in partially folded states. This can alter the interpretation of mutational protein engineering results in terms of native-like side chain interactions. In addition, since the intermediate exists after the rate-limiting step and Rd-apocyt b(562) folds very rapidly (k(f) approximately 10(4) s(-1)), these results suggest that non-native hydrophobic interactions, in the absence of topological misfolding, are repaired too rapidly to slow folding and cause the accumulation of folding intermediates. More generally, these results illustrate an approach for determining the high-resolution structure of folding intermediates. 相似文献
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The absence of detectable kinetic and equilibrium folding intermediates by optical probes is commonly taken to indicate that protein folding is a two-state process. However, for some small proteins with apparent two-state behavior, unfolding intermediates have been identified in native-state hydrogen exchange or kinetic unfolding experiments monitored by nuclear magnetic resonance. Rd-apocytochrome b(562), a four-helix bundle, is one such protein. Here, we found another unfolding intermediate for Rd-apocytochrome b(562). It is based on a cooperative transition of (15)N chemical shifts of amide protons as a function of urea concentrations before the global unfolding. We have solved the high-resolution structure of the protein at 2.8 M urea, which is after this cooperative transition but before the global unfolding. All four helices remained intact, but a number of hydrophobic core residues repacked. This intermediate provides a possible structural interpretation for the kinetic unfolding intermediates observed using nuclear magnetic resonance methods for several proteins and has important implications for theoretical studies of protein folding. 相似文献
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