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The larger protein complexes of the cyanobacterial photosynthetic membrane of Thermosynechoccus elongatus and Synechocystis 6803 were studied by single particle electron microscopy after detergent solubilization, without any purification steps. Besides the "standard" L-shaped NDH-1L complex, related to complex I, large numbers of a U-shaped NDH-1MS complex were found in both cyanobacteria. In membranes from Synechocystis DeltacupA and DeltacupA/cupB mutants the U-shaped complexes were absent, indicating that CupA is responsible for the U-shape by binding at the tip of the membrane-bound arm of NDH-1MS. Comparison of membranes grown under air levels of CO(2) or 3% CO(2) indicates that the number of NDH-1MS particles is 30-fold higher under low-CO(2). 相似文献
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The work presented here describes a new and simple method based on site-directed fluorescence labeling using the BADAN label that permits the examination of protein-lipid interactions in great detail. We applied this technique to a membrane-embedded, mainly α-helical reference protein, the M13 major coat protein. Using a high-throughput approach, 40 site-specific cysteine mutants were prepared of the 50-residues long protein. The steady-state fluorescence spectra were analyzed using a three-component spectral model that enabled the separation of Stokes shift contributions from water and internal label dynamics, and protein topology. We found that most of the fluorescence originated from BADAN labels that were hydrogen-bonded to water molecules even within the hydrophobic core of the membrane. Our spectral decomposition method revealed the embedment and topology of the labeled protein in the membrane bilayer under various conditions of headgroup charge and lipid chain length, as well as key characteristics of the membrane such as hydration level and local polarity, provided by the local dielectric constant. 相似文献
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Human α-N-acetylglucosaminidase (Naglu) is a lysosomal acid hydrolase implicated in Mucopolysaccharidosis type IIIB (MPS IIIB). We utilize a Spodoptera frugiperda (Sf9) system to express Naglu fused to a synthetic protein transduction domain in hopes to facilitate delivery of Naglu across the blood–brain barrier, thus allowing enzyme replacement therapy to treat neurological symptoms.Although human recombinant Naglu was previously produced in Sf9, low expression levels suggested degradation via cryptic mRNA splicing. Two cryptic splice sites discovered within Naglu cDNA were altered by site-directed mutagenesis, reducing Naglu mRNA degradation. A native Naglu secretion-signaling peptide was efficiently recognized by the Sf9 system. Significantly higher enzyme activity was seen from multiple adherent Sf9 cultures stably expressing mutagenized Naglu over those expressing wildtype Naglu (P = 0.000; 3.4-fold average increased specific activity). Suspension cultures demonstrated a 4.0-fold increase in overall enzyme activity secreted post-mutagenesis. Thus elimination of cryptic splicing directly resulted in higher Naglu expression. 相似文献
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《Molecular & cellular proteomics : MCP》2020,19(1):114-127
Highlights
- •Quantitative proteomics and machine learning to study plasma biomarkers in HCM.
- •Six peptides are increased in plasma of LVH+ HCM compared to controls.
- •Peptide biomarkers correlate with imaging markers of phenotype severity.
- •Peptide biomarkers correlate with the estimated sudden cardiac death risk.