Sedimentation, viscosity and partial specific volumes of membrane proteins and lipoproteins.
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ABSTRACT:
SUBMITTER: Brown AD
PROVIDER: S-EPMC1270363 | biostudies-other | 1967 Apr
REPOSITORIES: biostudies-other
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Project description:Accurate partial-specific volume ([Formula: see text]) values are required for sedimentation velocity and sedimentation equilibrium analyses. For nucleic acids, the estimation of these values is complicated by the fact that [Formula: see text] depends on base composition, secondary structure, solvation and the concentrations and identities of ions in the surrounding buffer. Here we describe sedimentation equilibrium measurements of the apparent isopotential partial-specific volume /' for two G-quadruplex DNAs and a single-stranded DNA of similar molecular weight and base composition. The G-quadruplex DNAs are a 22 nucleotide fragment of the human telomere consensus sequence and a 27 nucleotide fragment from the human c-myc promoter. The single-stranded DNA is 26 nucleotides long and is designed to have low propensity to form secondary structures. Parallel measurements were made in buffers containing NaCl and in buffers containing KCl, spanning the range 0.09 M <or= [salt] <or= 2.3 M. Limiting values of /', extrapolated to [salt] = 0 M, were: 22-mer (NaCl-form), 0.525 +/- 0.004 mL/g; 22-mer (KCl-form), 0.531 +/- 0.006 mL/g; 27-mer (NaCl-form), 0.548 +/- 0.005 mL/g; 27-mer (KCl-form), 0.557 +/- 0.006 mL/g; 26-mer (NaCl-form), 0.555 +/- 0.004 mL/g; 26-mer (KCl-form), 0.564 +/- 0.006 mL/g. Small changes /' with [salt] suggest that large changes in counterion association or hydration are unlikely to take place over these concentration ranges.
| S-EPMC2813399 | biostudies-literature
Project description:Background. Bacillus subtilis is a favorable host for the production of industrially relevant proteins because of its capacity of secreting proteins into the medium to high levels, its GRAS (Generally Recognized As Safe) status, its genetic accessibility and its capacity to grow in large fermentations. However, production of heterologous proteins still faces limitations. Results. This study aimed at the identification of bottlenecks in secretory protein production by analyzing the response of B. subtilis at the transcriptome level to overproduction of eight secretory proteins of endogenous and heterologous origin and with different subcellular or extracellular destination: secreted proteins (NprE and XynA of B. subtilis, Usp45 of Lactococcus lactis, TEM-1beta -lactamase of Escherichia coli), membrane proteins (LmrA of L. lactis and XylP of Lactobacillus pentosus) and lipoproteins (MntA and YcdH of B. subtilis). Responses specific for proteins with a common localization as well as more general stress responses were observed. The latter include upregulation of genes encoding intracellular stress proteins (groES/EL, CtsR regulated genes). Specific responses include upregulation of the liaIHGFSR operon under Usp45 and TEM-1 beta-lactamase overproduction; cssRS, htrA and htrB under all secreted proteins overproduction; sigW and SigW-regulated genes mainly under membrane proteins overproduction; and ykrL (encoding an HtpX homologue) specifically under membrane proteins overproduction. Conclusions. The results give better insights into B. subtilis responses to protein overproduction stress and provide potential targets for genetic engineering in order to further improve B. subtilis as a protein production host.
2012-05-25 | GSE34505 | GEO
Project description:Background. Bacillus subtilis is a favorable host for the production of industrially relevant proteins because of its capacity of secreting proteins into the medium to high levels, its GRAS (Generally Recognized As Safe) status, its genetic accessibility and its capacity to grow in large fermentations. However, production of heterologous proteins still faces limitations. Results. This study aimed at the identification of bottlenecks in secretory protein production by analyzing the response of B. subtilis at the transcriptome level to overproduction of eight secretory proteins of endogenous and heterologous origin and with different subcellular or extracellular destination: secreted proteins (NprE and XynA of B. subtilis, Usp45 of Lactococcus lactis, TEM-1beta -lactamase of Escherichia coli), membrane proteins (LmrA of L. lactis and XylP of Lactobacillus pentosus) and lipoproteins (MntA and YcdH of B. subtilis). Responses specific for proteins with a common localization as well as more general stress responses were observed. The latter include upregulation of genes encoding intracellular stress proteins (groES/EL, CtsR regulated genes). Specific responses include upregulation of the liaIHGFSR operon under Usp45 and TEM-1 beta-lactamase overproduction; cssRS, htrA and htrB under all secreted proteins overproduction; sigW and SigW-regulated genes mainly under membrane proteins overproduction; and ykrL (encoding an HtpX homologue) specifically under membrane proteins overproduction. Conclusions. The results give better insights into B. subtilis responses to protein overproduction stress and provide potential targets for genetic engineering in order to further improve B. subtilis as a protein production host. Samples for transcriptome analyses were induced at the exponential-growth phase (OD600 = 0.7) with 0.1% subtilin (subtilin containing supernatant of subtilin producing B. subtilis strain ATCC 6633). Cells were harvested 30 min after induction. Three or four independent cultures of each strain (target strains and controls) were used, and cells were sampled for microarray experiment.
2012-05-24 | E-GEOD-34505 | biostudies-arrayexpress
Project description:BACKGROUND: Bacillus subtilis is a favorable host for the production of industrially relevant proteins because of its capacity of secreting proteins into the medium to high levels, its GRAS (Generally Recognized As Safe) status, its genetic accessibility and its capacity to grow in large fermentations. However, production of heterologous proteins still faces limitations. RESULTS: This study aimed at the identification of bottlenecks in secretory protein production by analyzing the response of B. subtilis at the transcriptome level to overproduction of eight secretory proteins of endogenous and heterologous origin and with different subcellular or extracellular destination: secreted proteins (NprE and XynA of B. subtilis, Usp45 of Lactococcus lactis, TEM-1 ?-lactamase of Escherichia coli), membrane proteins (LmrA of L. lactis and XylP of Lactobacillus pentosus) and lipoproteins (MntA and YcdH of B. subtilis). Responses specific for proteins with a common localization as well as more general stress responses were observed. The latter include upregulation of genes encoding intracellular stress proteins (groES/EL, CtsR regulated genes). Specific responses include upregulation of the liaIHGFSR operon under Usp45 and TEM-1 ?-lactamase overproduction; cssRS, htrA and htrB under all secreted proteins overproduction; sigW and SigW-regulated genes mainly under membrane proteins overproduction; and ykrL (encoding an HtpX homologue) specifically under membrane proteins overproduction. CONCLUSIONS: The results give better insights into B. subtilis responses to protein overproduction stress and provide potential targets for genetic engineering in order to further improve B. subtilis as a protein production host.
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Project description:The deduced sequences of the Omp10, Omp16, and Omp19 outer membrane proteins of Brucella spp. contain a potential bacterial lipoprotein processing sequence. After extraction with Triton X-114, these three proteins partitioned into the detergent phase. Processing of the three proteins is inhibited by globomycin, a specific inhibitor of lipoprotein signal peptidase. The three proteins were radioimmunoprecipitated from [(3)H]palmitic acid-labeled Brucella abortus lysates with monoclonal antibodies. These results demonstrate that Omp10, Omp16, and Omp19 are lipoproteins.
| S-EPMC96837 | biostudies-literature
Project description:1. Sedimentation-velocity, intrinsic-viscosity and partial-specific-volume measurements on a typical blood-group-specific glycoprotein are reported for a range of environmental conditions. 2. The sedimentation coefficients, S, are strongly concentration-dependent, and follow the reciprocal law; the limiting values at 2 degrees , 25 degrees and 45 degrees , after correction to 25 degrees , show slight dependence on temperature. 3. The intrinsic viscosities, [eta], at 25 degrees and 45 degrees show more marked temperature-dependence, and correspond to a very asymmetric or very expanded molecular conformation. 4. From the value of the ratio K/[eta], where K=S(0).d(1/S)/dc, it is concluded that the molecular conformation is roughly spherical; application of the Einstein viscosity equation then suggests an expansion factor of about 60, compatible with a flexible configuration approaching that of a random coil. 5. The sedimentation coefficient is not affected by variation of ionic strength in the range 0.01-0.50, nor by pH in the range 3-10. 6. Sodium dodecyl sulphate at 1.5% produces a small decrease in S; the effect is greater than would be expected from the observed extent of binding, but is too small to correspond to a significant change in secondary structure; the serological activity is unaffected by sodium dodecyl sulphate. 7. All the properties observed indicate the absence of any secondary structure in blood-group substances.
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Project description:Partial molar volume is an important thermodynamic property that gives insights into molecular size and intermolecular interactions in solution. Theoretical frameworks for determining the partial molar volume (V°) of a solvated molecule generally apply Scaled Particle Theory or Kirkwood-Buff theory. With the current abilities to perform long molecular dynamics and Monte Carlo simulations, more direct methods are gaining popularity, such as computing V° directly as the difference in computed volume from two simulations, one with a solute present and another without. Thermodynamically, V° can also be determined as the pressure derivative of the free energy of solvation in the limit of infinite dilution. Both approaches are considered herein with the use of free energy perturbation (FEP) calculations to compute the necessary free energies of solvation at elevated pressures. Absolute and relative partial molar volumes are computed for benzene and benzene derivatives using the OPLS-AA force field. The mean unsigned error for all molecules is 2.8 cm(3) mol(-1). The present methodology should find use in many contexts such as the development and testing of force fields for use in computer simulations of organic and biomolecular systems, as a complement to related experimental studies, and to develop a deeper understanding of solute-solvent interactions.
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Project description:The erythrocyte sedimentation rate (ESR) is a non-specific blood test for determining inflammatory conditions. However, the long measurement time (60 min) to obtain ESR is an obstacle for a prompt evaluation. In this study, to reduce the measurement time of ESR, deep neural networks (DNNs) were applied to the sedimentation tendency of blood samples. DNNs using multilayer perceptron (MLP), long short-term memory (LSTM), and gated recurrent unit (GRU) were assessed and compared to determine a suitable length of time for the input sequence. To avoid overfitting, a stacking ensemble learning was adopted, which combines multiple models by using a meta model. Four meta models were compared: mean, median, least absolute shrinkage and selection operator, and partial least squares regression (PLSR) schemes. From the empirical results, LSTM and GRU models have better prediction than MLP over sequence lengths of 5 to 20 min. The decrease in [Formula: see text] and [Formula: see text] of GRU and LSTM was attenuated after a sequence length of 15 min, so the input sequence length is determined as 15 min. In terms of the meta model, the statistical comparison suggests that GRU combined with PLSR (GRU-PLSR) is the best case. Then, the GRU-PLSR was tested for prediction of ESR data obtained from periodontitis patients to check its applicability to a specific disease. The Bland-Altman plot shows acceptable agreement between measured and predicted ESR values. Based on the results, the GRU-PLSR can predict ESR with improved performance within 15 min and has potential applicability to ESR data with inflammatory and non-inflammatory conditions.
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