scholarly journals NMR studies reveal that protein dynamics critically mediate aggregation of the well-folded and very soluble E. coli S1 ribosomal protein

2017 ◽  
Author(s):  
Yimei Lu ◽  
Liangzhong Lim ◽  
Jianxing Song
Keyword(s):  
Fluorescence Spectroscopy ◽  
Protein Aggregation ◽  
Ribosomal Protein ◽  
Conformational Dynamics ◽  
Nmr Studies ◽  
E Coli ◽  
Domain Interactions ◽  
Irreversible Unfolding ◽  
The Relationship

AbstractUnlike mammalian aging associated with many hallmarks, E. coli aging is only significantly characterized by protein aggregation, thus offering an excellent model for addressing the relationship between protein aggregation and aging. Here we characterized conformations, unfolding and dynamics of ribosomal protein S1 and its D3/D5 domains using NMR, CD and fluorescence spectroscopy. S1 is a 557-residue modular protein containing six S1 motifs. Paradoxically, while S1 is well-folded and very soluble in vitro, it was found in various lists of aggregated E. coli proteins. Our results decipher: 1) S1 has dynamic inter-domain interactions. Strikingly, S1 and its D3/D5 domains have significantly exposed hydrophobic patches characterized by irreversible unfolding. 2) Although D5 has significantly restricted backbone motion on ps-ns time scale, it has global μs-ms conformational dynamics and particularly high “global breathing” motions. 3) D5 assumes the conserved β-barrel fold but contains large hydrophobic patches at least dynamically accessible. Taken together, our study reveals that S1 could be prone to aggregation due to significant dynamics at two levels: inter-domain interactions and individual domains, which may even render buried hydrophobic patches/cores accessible for driving aggregation. This mechanism is most likely to operate in many proteins of E. coli and other organisms including human.

Expression, purification and properties of multidrug efflux proteins

2000 ◽  
Vol 28 (4) ◽  
pp. 513-517 ◽  
Author(s):  
P.J. F. Henderson ◽  
C. K. Hoyle ◽  
A. Ward
Keyword(s):  
Fluorescent Protein ◽  
Affinity Column ◽  
General Strategy ◽  
X Rays ◽  
Multidrug Efflux ◽  
Nmr Studies ◽  
E Coli ◽  
Efflux Proteins

A general strategy is described for the amplified expression, purification and characterization in Escherichia coli of multidrug efflux proteins from Staphylococcus aureus, Bacillus subtilis, Methanococcus janaschii and E. coli. They all catalyse drug/H+ antiport of substrates such as quinolones and ethidium and exemplify a family of putatively 12-helix membrane proteins. The gene for each protein was cloned downstream of the tac promoter in plasmid pTTQ18; an oligonucleotide encoding six histidine residues was added, in frame, to the C-terminus to facilitate purification. Growth conditions were optimized in 1–25-litre cultures of E. coli host strains to amplify the expression of each protein; the retention of activity was confirmed by assays of antibiotic resistance in vivo and/or assays of energized transport activity in vitro with synthetic substrates. Proteins were solubilized in dodecylmaltoside and purified to more than 90% homogeneity with Ni2+-nitrilo-triacetate-affinity column chromography, yielding 5–25 mg per 25 litres of original culture. All the transport proteins migrated anomalously in SDS/PAGE at apparent molecular masses below those predicted from the gene sequence; identity and integrity were therefore confirmed by N-terminal amino acid sequencing and Western blotting for the C-terminal hexahistidine tag. Examination of the secondary structure of detergent-solubilized proteins by CD or Fourier-transform infrared spectroscopy following purification indicated a high content of α-helix (more than 75%). Matrix-assisted laser desorption ionization MS confirmed the high degree of purity and the true molecular mass. The formation of three-dimensional crystals is being attempted but crystals have yet to be grown that diffract X-rays. The growth of two-dimensional protein arrays has been more successful, with diffraction of electrons at low resolution. Proteins have been fused to green fluorescent protein or maltose-binding protein to facilitate these structural analyses. In addition, ligands for efflux proteins labelled with 13C or 15N have been synthesized to implement solid-state NMR studies of the ligand-binding site.

The relationship between the spoT gene, the synthesis of stable RNA, ribosomal proteins, and the ββ′ subunits of RNA polymerase following a nutritional shiftup of Escherichia coli

1978 ◽  
Vol 56 (6) ◽  
pp. 528-533 ◽  
Author(s):  
Stephen M. Boyle ◽  
Frederick Chu ◽  
Nathan Brot ◽  
Bruce H. Sells
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Rna Synthesis ◽  
Transient Increase ◽  
Spot Gene ◽  
The Relationship

The level of ppGpp and rates of synthesis of stable RNA, ribosomal protein, and the β and β′ subunits of RNA polymerase were measured following a nutritional shiftup in Escherichia coli strains, NF 929 (spoT+) and NF 930 (spoT'−). In the spoT+ strain, ppGpp levels decreased 50% within 2 min following shiftup, and the rates of synthesis of stable RNA, ribosomal proteins, and the β and β′ subunits of RNA polymerase increased with little or no lag. In contrast, in the spoT− strain, ppGpp levels transiently increased 40% during the first 6 min following shiftup. An inhibition in the rate of stable RNA synthesis and a delay in the increased synthesis of ribosomal proteins and β and β′ subunits occurred concurrently with the transient increase in ppGpp. In addition, the DNA-dependent synthesis in vitro of the β and β′ subunits of RNA polymerase was inhibited by physiological levels of ppGpp. Because of the timing and magnitude of the changes in ppGpp levels in the spoT− strain versus the timing when the new rates of stable RNA, ribosomal protein, and β and β′ subunits synthesis are reached, it is concluded that ppGpp is not the sole element regulating the expression of these genes.

scholarly journals The Chemical Chaperone Proline Relieves the Thermosensitivity of a dnaK Deletion Mutant at 42°C

2004 ◽  
Vol 186 (23) ◽  
pp. 8149-8152 ◽  
Author(s):  
Madhab K. Chattopadhyay ◽  
Renée Kern ◽  
Michel-Yves Mistou ◽  
Abhaya M. Dandekar ◽  
Sandra L. Uratsu ◽  
...  
Keyword(s):  
Protein Aggregation ◽  
Citrate Synthase ◽  
Feedback Inhibition ◽  
Single Mutation ◽  
Two Dimensional Gel Electrophoresis ◽  
Chemical Chaperone ◽  
E Coli ◽  
Amino Acid Biosynthetic Pathway

ABSTRACT Since, like other osmolytes, proline can act as a protein stabilizer, we investigated the thermoprotectant properties of proline in vitro and in vivo. In vivo, elevated proline pools in Escherichia coli (obtained by altering the feedback inhibition by proline of γ-glutamylkinase, the first enzyme of the proline biosynthesis pathway) restore the viability of a dnaK-deficient mutant at 42°C, suggesting that proline can act as a thermoprotectant for E. coli cells. Furthermore, analysis of aggregated proteins in the dnaK-deficient strain at 42°C by two-dimensional gel electrophoresis shows that high proline pools reduce the protein aggregation defect of the dnaK-deficient strain. In vitro, like other “chemical chaperones,” and like the DnaK chaperone, proline protects citrate synthase against thermodenaturation and stimulates citrate synthase renaturation after urea denaturation. These results show that a protein aggregation defect can be compensated for by a single mutation in an amino acid biosynthetic pathway and that an ubiquitously producible chemical chaperone can compensate for a defect in one of the major chaperones involved in protein folding and aggregation.

scholarly journals Pharmacological Basis of β-Lactamase Inhibitor Therapeutics: Tazobactam in Combination with Ceftolozane

2013 ◽  
Vol 57 (12) ◽  
pp. 5924-5930 ◽  
Author(s):  
Brian VanScoy ◽  
Rodrigo E. Mendes ◽  
Jennifer McCauley ◽  
Sujata M. Bhavnani ◽  
Catharine C. Bulik ◽  
...  
Keyword(s):  
Threshold Concentration ◽  
Genetically Engineered ◽  
Clinical Isolates ◽  
Enzyme Expression ◽  
Data Set ◽  
Content Type ◽  
E Coli ◽  
Resistance Determinants ◽  
The Relationship

ABSTRACTWe recently investigated the pharmacokinetics-pharmacodynamics (PK-PD) of tazobactam in combination with ceftolozane against an isogenic CTX-M-15-producingEscherichia colitriplet set, genetically engineered to transcribe different levels ofblaCTX-M-15. The percentage of the dosing interval that tazobactam concentrations remained above a threshold (%Time>threshold) was identified as the PK-PD exposure measure that was most closely associated with efficacy. Moreover, the tazobactam concentration was dependent upon the enzyme transcription level. Given that the aforementioned strains were genetically engineered to transcribe a single β-lactamase enzyme and that clinical isolates typically produce multiple β-lactamase enzymes with various transcription levels, it is likely that the tazobactam threshold concentration is isolate/enzyme dependent. Our first objective was to characterize the relationship between the tazobactam %Time>threshold in combination with ceftolozane and efficacy using clinical isolates in anin vitroPK-PD infection model. Our second objective was to identify a translational relationship that would allow for the comodeling across clinical isolates. The initial challenge panel included four well-characterized β-lactamase-producingE. colistrains with variable enzyme expression and other resistance determinants. As evidenced byr2values of ranging from 0.90 to 0.99 for each clinical isolate, the observed data were well described by fitted functions describing the relationship between the tazobactam %Time>threshold and change in log10CFU from baseline; however, the data from the four isolates did not comodel well. The threshold concentration identified for each isolate ranged from 0.5 to 4 mg/liter. We identified an enabling translational relationship for the tazobactam threshold that allowed comodeling of all four clinical isolates, which was the product of the individual isolate's ceftolozane-tazobactam MIC value and 0.5. As evidenced by anr2value of 0.90, the transformed data were well described by a fitted function describing the relationship between tazobactam %Time>threshold and change in log10CFU from baseline. Due to these findings, the challenge panel was expanded to include three well-characterized β-lactamase-producingKlebsiella pneumoniaestrains with variable enzyme expression and other resistance determinants. The translational relationship for the tazobactam threshold that allowed for the comodeling of the fourE. coliisolates performed well for the expanded data set (seven isolates in total; fourE. coliand threeK. pneumoniae), as evidenced by anr2value of 0.84. This simple translational relationship is especially useful as it is directly linked toin vitrosusceptibility test results, which are used to guide the clinician's choice of drug and dosing regimen.

scholarly journals Antibiotic resistance in Escherichia coli causing generalized infections in chickens in the UK in 1982: the relationship between the results of in vitro and in vivo furazolidone sensitivity tests

1984 ◽  
Vol 93 (3) ◽  
pp. 445-453 ◽  
Author(s):  
H. Williams Smith ◽  
M. A. Lovell
Keyword(s):  
Escherichia Coli ◽  
Laboratory Tests ◽  
Tetracycline Resistance ◽  
E Coli ◽  
Sensitivity Tests ◽  
Similar Survey ◽  
The Uk ◽  
The Relationship

SummaryCompared with a similar survey conducted ten years previously, a survey conducted in 1982, eleven years after the implementation of legislation forbidding the routine use of feeds containing ‘therapeutic’ antibiotics, revealed a decreased incidence of resistance to tetracyclines, furazolidone and sulphonamides in Escherichia coli strains causing generalized infections in chickens in the UK; the decrease was particularly marked in the case of tetracycline resistance, 17·9% of strains in 1982 being resistant to this antibiotic compared with 31·2% in 1972.Giving furazolidone to groups of chickens inoculated intramuscularly with O2:K1 strains of E. coli of differing degrees of furazolidone sensitivity indicated that great care is required in the performance and interpretation of laboratory tests for sensitivity to this antibiotic. Infections caused by strains that required as little as 1·25 μg/ml of furazolidone to inhibit their multiplication in laboratory tests responded poorly to furazolidone treatment; those that were inhibited by less responded well, better than to treatment with tetrac3rcline, chloramphenicol. ampicillin or trimethoprim.

scholarly journals The stoichiometry of E. coli 30S ribosomal protein S1 on in vivo and in vitro polyribosomes

FEBS Letters ◽  
1974 ◽  
Vol 41 (2) ◽  
pp. 323-326 ◽  
Author(s):  
P.H. Van Knippenberg ◽  
P.J.J. Hooykaas ◽  
J. Van Duin
Keyword(s):  
Ribosomal Protein ◽  
E Coli ◽  
Ribosomal Protein S1

scholarly journals Thermus thermophilus L11 Methyltransferase, PrmA, Is Dispensable for Growth and Preferentially Modifies Free Ribosomal Protein L11 Prior to Ribosome Assembly

2004 ◽  
Vol 186 (17) ◽  
pp. 5819-5825 ◽  
Author(s):  
Dale M. Cameron ◽  
Steven T. Gregory ◽  
Jill Thompson ◽  
Moo-Jin Suh ◽  
Patrick A. Limbach ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Thermus Thermophilus ◽  
Methyl Groups ◽  
Maldi Tof Ms ◽  
E Coli ◽  
Maldi Tof ◽  
Ribosomal Protein L11 ◽  
Tof Ms

ABSTRACT The ribosomal protein L11 in bacteria is posttranslationally trimethylated at multiple amino acid positions by the L11 methyltransferase PrmA, the product of the prmA gene. The role of L11 methylation in ribosome function or assembly has yet to be determined, although the deletion of Escherichia coli prmA has no apparent phenotype. We have constructed a mutant of the extreme thermophile Thermus thermophilus in which the prmA gene has been disrupted with the htk gene encoding a heat-stable kanamycin adenyltransferase. This mutant shows no growth defects, indicating that T. thermophilus PrmA, like its E. coli homolog, is dispensable. Ribosomes prepared from this mutant contain unmethylated L11, as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and are effective substrates for in vitro methylation by cloned and purified T. thermophilus PrmA. MALDI-TOF MS also revealed that T. thermophilus L11 contains a total of 12 methyl groups, in contrast to the 9 methyl groups found in E. coli L11. Finally, we found that, as with the E. coli methyltransferase, the ribosomal protein L11 dissociated from ribosomes is a more efficient substrate for in vitro methylation by PrmA than intact 70S ribosomes, suggesting that methylation in vivo occurs on free L11 prior to its incorporation into ribosomes.

scholarly journals Insight into the molecular mechanism of miR-192 regulating Escherichia coli resistance in piglets

2018 ◽  
Vol 38 (1) ◽  
Author(s):  
Li Sun ◽  
Sen Wu ◽  
Chao-Hui Dai ◽  
Shou-Yong Sun ◽  
Guo-Qiang Zhu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanism ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Transcription Activator ◽  
E Coli ◽  
Cellular Processes ◽  
Adhesion Ability ◽  
The Relationship

MicroRNAs (miRNAs) have important roles in many cellular processes, including cell proliferation, growth and development, and disease control. Previous study demonstrated that the expression of two highly homologous miRNAs (miR-192 and miR-215) was up-regulated in weaned piglets with Escherichia coli F18 infection. However, the potential molecular mechanism of miR-192 in regulating E. coli infection remains unclear in pigs. In the present study, we analyzed the relationship between level of miR-192 and degree of E. coli resistance using transcription activator-like effector nuclease (TALEN), in vitro bacterial adhesion assays, and target genes research. A TALEN expression vector that specifically recognizes the pig miR-192 was constructed and then monoclonal epithelial cells defective in miR-192 were established. We found that miR-192 knockout led to enhance the adhesion ability of the E. coli strains F18ab, F18ac and K88ac, meanwhile increase the expression of target genes (DLG5 and ALCAM) by qPCR and Western blotting analysis. The results suggested that miR-192 and its key target genes (DLG5 and ALCAM) could have a key role in E. coli infection. Based on our findings, we propose that further investigation of miR-192 function is likely to lead to insights into the molecular mechanisms of E. coli infection.

scholarly journals RACK1 on and off the ribosome

2019 ◽  
Author(s):  
Alex G. Johnson ◽  
Christopher P. Lapointe ◽  
Jinfan Wang ◽  
Nicholas C. Corsepius ◽  
Junhong Choi ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Ribosomal Subunit ◽  
Fluorescent Labeling ◽  
Cellular Functions ◽  
Biochemical Basis ◽  
Biochemical Analyses ◽  
Single Molecule Analysis

ABSTRACTReceptor for activated C kinase 1 (RACK1) is a eukaryote-specific ribosomal protein implicated in diverse biological functions. To engineer ribosomes for specific fluorescent labeling, we selected RACK1 as an target given its location on the small ribosomal subunit and other properties. However, prior results suggested that RACK1 has roles both on and off the ribosome, and such an exchange might be related to its various cellular functions and hinder our ability to use RACK1 as a stable fluorescent tag for the ribosome. In addition, the kinetics of spontaneous exchange of RACK1 or any ribosomal protein from a mature ribosomein vitroremain unclear. To address these issues, we engineered fluorescently-labeled human ribosomes via RACK1, and applied bulk and single-molecule biochemical analyses to track RACK1 on and off the human ribosome. Our results demonstrate that, despite its cellular non-essentiality from yeast to humans, RACK1 readily re-associates with the ribosome, displays limited conformational dynamics, and remains stably bound to the ribosome for hoursin vitro. This work sheds insight onto the biochemical basis of ribosomal protein exchange on and off a mature ribosome and provides tools for single-molecule analysis of human translation.

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