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11.
《Journal of molecular biology》2021,433(15):167098
MPV17 is an integral inner mitochondrial membrane protein, whose loss-of-function is linked to the hepatocerebral form of the mitochondrial-DNA-depletion syndrome, leading to a tissue-specific reduction of mitochondrial DNA and organ failure in infants. Several disease-causing mutations in MPV17 have been identified and earlier studies with reconstituted protein suggest that MPV17 forms a high conductivity channel in the membrane. However, the molecular and structural basis of the MPV17 functionality remain only poorly understood. In order to make MPV17 accessible to high-resolution structural studies, we here present an efficient protocol for its high-level production in E. coli and refolding into detergent micelles. Using biophysical and NMR methods, we show that refolded MPV17 in detergent micelles adopts a compact structure consisting of six membrane-embedded α-helices. Furthermore, we demonstrate that MPV17 forms oligomers in a lipid bilayer that are further stabilized by disulfide-bridges. In line with these findings, MPV17 could only be inserted into lipid nanodiscs of 8–12 nm in diameter if intrinsic cysteines were either removed by mutagenesis or blocked by chemical modification. Using this nanodisc reconstitution approach, we could show that disease-linked mutations in MPV17 abolish its oligomerization properties in the membrane. These data suggest that, induced by oxidative stress, MPV17 can alter its oligomeric state from a properly folded monomer to a disulfide-stabilized oligomeric pore which might be required for the transport of metabolic DNA precursors into the mitochondrial matrix to compensate for the damage caused by reactive oxygen species. 相似文献
12.
《Journal of molecular biology》2021,433(19):167189
Primase is an essential component of the DNA replication machinery, responsible for synthesizing RNA primers that initiate leading and lagging strand DNA synthesis. Bacterial primase activity can be regulated by the starvation-inducible nucleotide (p)ppGpp. This regulation contributes to a timely inhibition of DNA replication upon amino acid starvation in the Gram-positive bacterium Bacillus subtilis. Here, we characterize the effect of (p)ppGpp on B. subtilis DnaG primase activity in vitro. Using a single-nucleotide resolution primase assay, we dissected the effect of ppGpp on the initiation, extension, and fidelity of B. subtilis primase. We found that ppGpp has a mild effect on initiation, but strongly inhibits primer extension and reduces primase processivity, promoting termination of primer extension. High (p)ppGpp concentration, together with low GTP concentration, additively inhibit primase activity. This explains the strong inhibition of replication elongation during starvation which induces high levels of (p)ppGpp and depletion of GTP in B. subtilis. Finally, we found that lowering GTP concentration results in mismatches in primer base pairing that allow priming readthrough, and that ppGpp reduces readthrough to protect priming fidelity. These results highlight the importance of (p)ppGpp in protecting replisome integrity and genome stability in fluctuating nucleotide concentrations upon onset of environmental stress. 相似文献
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J.C. Smith R.B. Derbyshire E. Cook L. Dunthorne J. Viney S.J. Brewer H.M. Sassenfeld L.D. Bell 《Gene》1984,32(3):321-327
A 43-bp DNA duplex coding for poly(arginine) [poly(arg)] has been synthesised by modified phosphotriester procedures. It has been inserted into the BglII and BamHI restriction sites of a cloned synthetic β-urogastrone (Uro) gene, under the control of the trp promoter. Subsequent induction with 3β-indole acrylic acid produces β-Uro with a C-terminal poly(arg) fusion. The raised isoelectric point of this polypeptide fusion facilitates rapid purification by cation exchange chromatography. The C-terminal poly(arg) tail can be readily removed by treatment with carboxypeptidase B. 相似文献
16.
C. J. Gray 《Biocatalysis and Biotransformation》1988,1(3):187-196
The polypeptide chain of an enzyme is folded so that the necessary functional groups are brought together in the active site. Conformational changes may disrupt this arrangement and cause loss of enzymic activity. The effect of soluble additives on the unfolding process is discussed. Additives may be classified as substrates and similar ligands, small uncharged organic molecules, specific and non-specific ionic species, and polymers. 相似文献
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Several metrics from nonlinear dynamics and statistical mechanics have been characterized on computer-generated number series with various signal-to-noise ratios, demonstrating their individual reliability as a function of sample size and their relationships to each other. The root mean square (RMS) evaluates amplitude, and the power spectral density (PSD) provides a visual display of the frequency spectrum; both measures have very high reliability even for an N as low as 50. The Fractal Dimension (D) is shown to converge rapidly and also to be reliable when N is as low as 50. These three measures (RMS, PSD, and D) have been applied to the complex kinetics of tyrosine hydroxylase time courses (50-point curves) at various BH4 concentrations (near physiological, but far from equilibrium levels). Recently developed measures of spectral entropy and the Liapunov Exponent, -lambda are also characterized. 相似文献
18.
We carried out an ecosystem service (ES) assessment at Soto de Pajares, a 400 ha active gravel quarry site located close to protected areas in the southeast of the Community of Madrid, Spain. The currently approved restoration plan is for quarry‐made excavations to be restored back to agricultural land. However, the site has been identified as being important for nature, so different restoration strategies should be considered. To better understand how these compare in terms of ES provision, which had not previously been done at mineral extraction sites in Spain, we used an established toolkit: the Toolkit for Ecosystem Service Site‐based Assessments (TESSA). We compared the agriculture‐focused restoration plan and two nature‐focused alternative scenarios, one without public access (conservation scenario) and the other with public access (compromise scenario). Monetary estimations of the value of agricultural production, climate change mitigation, and recreation were calculated in three scenarios. Results indicated that the compromise scenario provided the greatest annual value (€91,409), mainly due to its potential visitors, surpassing both agricultural (€68,504) and conservation (€48,556) scenarios. Considering the higher costs associated with restoring sites to agricultural production, nature conservation may be an attractive option for extraction companies. The use of TESSA at this site has provided valuable information to quarry managers to help guide their decision‐making. 相似文献
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This study evaluated the gait stability, variability, and complexity of healthy young adults on inclined surfaces. A total of 49 individuals walked on a treadmill at their preferred speed for 4 min at inclinations of 6%, 8%, and 10% in upward (UP) and downward (DOWN) conditions, and in horizontal (0%) condition. Gait variability was assessed using average standard deviation trunk acceleration between strides (VAR), gait stability was assessed using margin of stability (MoS) and maximum Lyapunov exponent (λs), and gait complexity was assessed using sample entropy (SEn). Trunk variability (VAR) increased in the medial-lateral (ML), anterior-posterior, and vertical directions for all inclined conditions. The SEn values indicated that movement complexity decreased almost linearly from DOWN to UP conditions, reflecting changes in gait pattern with longer and slower steps as inclination increased. The DOWN conditions were associated with the highest variability and lowest stability in the MoS ML, but not in λs. Stability was lower in UP conditions, which exhibited the largest λs values. The overall results support the hypothesis that inclined surfaces decrease gait stability and alter gait variability, particularly in UP conditions. 相似文献