scholarly journals Nucleotide binding to human uncoupling protein-2 refolded from bacterial inclusion bodies

2002 ◽  
Vol 366 (2) ◽  
pp. 565-571 ◽  
Author(s):  
Mika B. JEKABSONS ◽  
Karim S. ECHTAY ◽  
Martin D. BRAND
Keyword(s):  
Inclusion Bodies ◽  
Dissociation Constants ◽  
Uncoupling Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Uncoupling Protein 2 ◽  
Pyrimidine Nucleoside ◽  
Kidney Mitochondria ◽  
Nucleoside Triphosphates ◽  
Order Of Magnitude

Experiments were performed to test the hypothesis that recombinant human uncoupling protein-2 (UCP2) ectopically expressed in bacterial inclusion bodies binds nucleotides in a manner identical with the nucleotide-inhibited uncoupling that is observed in kidney mitochondria. For this, sarkosyl-solubilized UCP2 inclusion bodies were treated with the polyoxyethylene ether detergent C12E9 and hydroxyapatite. Protein recovered from hydroxyapatite chromatography was approx. 90% pure UCP2, as judged by Coomassie Blue and silver staining of polyacrylamide gels. Using fluorescence resonance energy transfer, N-methylanthraniloyl-tagged purine nucleoside di- and tri-phosphates exhibited enhanced fluorescence with purified UCP2. Dissociation constants determined by least-squares non-linear regression indicated that the affinity of UCP2 for these fluorescently tagged nucleotides was 3–5μM or perhaps an order of magnitude stronger, depending on the model used. Competition experiments with [8-14C]ATP demonstrated that UCP2 binds unmodified purine and pyrimidine nucleoside triphosphates with 2–5μM affinity. Affinities for ADP and GDP were approx. 10-fold lower. These data indicate that: UCP2 (a) is at least partially refolded from sarkosyl-solubilized bacterial inclusion bodies by a two-step treatment with C12E9 detergent and hydroxyapatite; (b) binds purine and pyrimidine nucleoside triphosphates with low micromolar affinity; (c) binds GDP with the same affinity as GDP inhibits superoxide-stimulated uncoupling by kidney mitochondria; and (d) exhibits a different nucleotide preference than kidney mitochondria.

scholarly journals Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators

2009 ◽  
Vol 106 (37) ◽  
pp. 15651-15656 ◽  
Author(s):  
Hiromi Imamura ◽  
Kim P. Huynh Nhat ◽  
Hiroko Togawa ◽  
Kenta Saito ◽  
Ryota Iino ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Atp Synthase ◽  
Hela Cells ◽  
Dissociation Constants ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Atp Levels ◽  
Fluorescence Resonance

Adenosine 5′-triphosphate (ATP) is the major energy currency of cells and is involved in many cellular processes. However, there is no method for real-time monitoring of ATP levels inside individual living cells. To visualize ATP levels, we generated a series of fluorescence resonance energy transfer (FRET)-based indicators for ATP that were composed of the ε subunit of the bacterial FoF1-ATP synthase sandwiched by the cyan- and yellow-fluorescent proteins. The indicators, named ATeams, had apparent dissociation constants for ATP ranging from 7.4 μM to 3.3 mM. By targeting ATeams to different subcellular compartments, we unexpectedly found that ATP levels in the mitochondrial matrix of HeLa cells are significantly lower than those of cytoplasm and nucleus. We also succeeded in measuring changes in the ATP level inside single HeLa cells after treatment with inhibitors of glycolysis and/or oxidative phosphorylation, revealing that glycolysis is the major ATP-generating pathway of the cells grown in glucose-rich medium. This was also confirmed by an experiment using oligomycin A, an inhibitor of FoF1-ATP synthase. In addition, it was demonstrated that HeLa cells change ATP-generating pathway in response to changes of nutrition in the environment.

scholarly journals Bioluminescent Ratiometric Indicator for Analysis of Water Hardness in Household Water

Sensors ◽  
2020 ◽  
Vol 20 (11) ◽  
pp. 3164
Author(s):  
Md Nadim Hossain ◽  
Ryuichi Ishida ◽  
Mitsuru Hattori ◽  
Tomoki Matsuda ◽  
Takeharu Nagai
Keyword(s):  
Fluorescent Protein ◽  
Dissociation Constants ◽  
Negative Impact ◽  
Low Cost ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Water Hardness ◽  
Hard Water ◽  
Household Water

Water hardness (WH) is a useful parameter for testing household water, such as drinking, cooking, and washing water. Many countries around the world use pipeline water in their houses, but there is a need to monitor the WH because hard water has a negative impact on appliances. Currently, WH is often measured using chemical dye-based WH indicators, and these techniques require expensive equipment, and trained personnel. Therefore, a low-cost and simple measurement method has been desired. Here, we report LOTUS-W, which consists of a luciferase, Nanoluc, a yellow fluorescent protein Venus, and a Ca2+/Mg2+ detection domain of human centrin 3. The binding of Ca2+/Mg2+ to this indicator changes the conformation of human centrin 3, and induces bioluminescence resonance energy transfer (BRET) from Nanoluc to Venus, which changes its emission spectrum about 140%. The dissociation constants of LOTUS-W for Ca2+/Mg2+ are approximately several mM, making it suitable for measuring WH in the household water. With this indicator in combination with a smartphone, we have demonstrated that it is possible to evaluate WH easily and quickly. This novel indicator has the potential to be used for measuring not only household water but also water used in the food industry, etc.

scholarly journals Secondary-structure characterization by far-UV CD of highly purified uncoupling protein 1 expressed in yeast

2004 ◽  
Vol 380 (1) ◽  
pp. 139-145 ◽  
Author(s):  
Pierre DOUETTE ◽  
Rachel NAVET ◽  
Fabrice BOUILLENNE ◽  
Alain BRANS ◽  
Claudine SLUSE-GOFFART ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Metal Ion ◽  
Binding Capacity ◽  
Uncoupling Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Homology Model ◽  
Structure Characterization ◽  
Uncoupling Protein 1 ◽  
Functional Protein

The rat UCP1 (uncoupling protein 1) is a mitochondrial inner-membrane carrier involved in energy dissipation and heat production. We expressed UCP1 carrying a His6 epitope at its C-terminus in Saccharomyces cerevisiae mitochondria. The recombinant-tagged UCP1 was purified by immobilized metal-ion affinity chromatography to homogeneity (>95%). This made it suitable for subsequent biophysical characterization. Fluorescence resonance energy transfer experiments showed that n-dodecyl-β-d-maltoside-solubilized UCP1–His6 retained its PN (purine nucleotide)-binding capacity. The far-UV CD spectrum of the functional protein clearly indicated the predominance of α-helices in the UCP1 secondary structure. The UCP1 secondary structure exhibited an α-helical degree of approx. 68%, which is at least 25% higher than the previously reported estimations based on computational predictions. Moreover, the helical content remained unchanged in free and PN-loaded UCP1. A homology model of the first repeat of UCP1, built on the basis of X-ray-solved close parent, the ADP/ATP carrier, strengthened the CD experimental results. Our experimental and computational results indicate that (i) α-helices are the major component of UCP1 secondary structure; (ii) PN-binding mechanism does not involve significant secondary-structure rearrangement; and (iii) UCP1 shares similar secondary-structure characteristics with the ADP/ATP carrier, at least for the first repeat.

Coenzyme Q induces GDP-sensitive proton conductance in kidney mitochondria

2001 ◽  
Vol 29 (6) ◽  
pp. 763-768 ◽  
Author(s):  
K. S. Echtay ◽  
M. D. Brand
Keyword(s):  
Superoxide Dismutase ◽  
Fatty Acid ◽  
Coenzyme Q10 ◽  
Redox State ◽  
Uncoupling Protein ◽  
Coenzyme Q ◽  
Liver Mitochondria ◽  
Uncoupling Protein 2 ◽  
Proton Conductance ◽  
Kidney Mitochondria

Addition of coenzyme Q10 (CoQ) at low concentration (29 nmol/mg of protein) to kidney but not liver mitochondria resulted in an increase in proton conductance. This uncoupling activity required fatty acid and was completely inhibited by GDP. CoQ activated when it was likely to be reduced but not when it was likely to become oxidized. However, the redox state of endogenous CoQ did not affect mitochondrial proton conductance. Stimulation by CoQ was not inhibited by cyclosporin A, carboxyatractylate, bongkrekate and catalase but could be reversed by superoxide dismutase. We conclude that CoQ acted in mitochondria through production of superoxide, which mediated uncoupling, probably by acting through uncoupling protein 2.

scholarly journals Polymer effects modulate binding affinities in disordered proteins

2019 ◽  
Vol 116 (39) ◽  
pp. 19506-19512 ◽  
Author(s):  
Renee Vancraenenbroeck ◽  
Yair S. Harel ◽  
Wenwei Zheng ◽  
Hagen Hofmann
Keyword(s):  
Single Molecule ◽  
Numerical Solutions ◽  
Regulatory Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Structural Disorder ◽  
Disordered Proteins ◽  
Binding Affinities ◽  
Order Of Magnitude ◽  
Functional Relevance

Structural disorder is widespread in regulatory protein networks. Weak and transient interactions render disordered proteins particularly sensitive to fluctuations in solution conditions such as ion and crowder concentrations. How this sensitivity alters folding coupled binding reactions, however, has not been fully understood. Here, we demonstrate that salt jointly modulates polymer properties and binding affinities of 5 disordered proteins from a transcription factor network. A combination of single-molecule Förster resonance energy transfer experiments, polymer theory, and molecular simulations shows that all 5 proteins expand with increasing ionic strengths due to Debye–Hückel charge screening. Simultaneously, pairwise affinities between the proteins increase by an order of magnitude within physiological salt limits. A quantitative analysis shows that 50% of the affinity increase can be explained by changes in the disordered state. Disordered state properties therefore have a functional relevance even if these states are not directly involved in biological functions. Numerical solutions of coupled binding equilibria with our results show that networks of homologous disordered proteins can function surprisingly robustly in fluctuating cellular environments, despite the sensitivity of its individual proteins.

scholarly journals Renal mitochondrial impairment is attenuated by AT1 blockade in experimental Type I diabetes

2008 ◽  
Vol 294 (1) ◽  
pp. H456-H465 ◽  
Author(s):  
Elena M. V. de Cavanagh ◽  
León Ferder ◽  
Jorge E. Toblli ◽  
Bárbara Piotrkowski ◽  
Inés Stella ◽  
...  
Keyword(s):  
Production Rate ◽  
Uncoupling Protein ◽  
The Other ◽  
Uncoupling Protein 2 ◽  
Control Groups ◽  
Kidney Mitochondria ◽  
Renal Lesions ◽  
And Control ◽  
Electron Transfer Capacity

To investigate whether ANG II type 1 (AT1) receptor blockade could protect kidney mitochondria in streptozotocin-induced Type 1 diabetes, we treated 8-wk-old male Sprague-Dawley rats with a single streptozotocin injection (65 mg/kg ip; STZ group), streptozotocin and drinking water containing either losartan (30 mg·kg−1·day−1; STZ+Los group) or amlodipine (3 mg·kg−1·day−1; STZ+Amlo group), or saline (intraperitoneally) and pure water (control group). Four-month-long losartan or amlodipine treatments started 30 days before streptozotocin injection to improve the antioxidant defenses. The number of renal lesions, plasma glucose and lipid levels, and proteinuria were higher and creatinine clearance was lower in STZ and STZ+Amlo compared with STZ+Los and control groups. Glycemia was higher in STZ+Los compared with control. Blood pressure, basal mitochondrial membrane potential and mitochondrial pyruvate content, and renal oxidized glutathione levels were higher and NADH/cytochrome c oxidoreductase activity was lower in STZ compared with the other groups. In STZ and STZ+Amlo groups, mitochondrial H2O2 production rate was higher and uncoupling protein-2 content, cytochrome c oxidase activity, and renal glutathione level were lower than in STZ+Los and control groups. Mitochondrial nitric oxide synthase activity was higher in STZ+Amlo compared with the other groups. Mitochondrial pyruvate content and H2O2 production rate negatively contributed to electron transfer capacity and positively contributed to renal lesions. Uncoupling protein-2 content negatively contributed to mitochondrial H2O2 production rate and renal lesions. Renal glutathione reduction potential positively contributed to mitochondria electron transfer capacity. In conclusion, AT1 blockade protects kidney mitochondria and kidney structure in streptozotocin-induced diabetes independently of blood pressure and glycemia.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

Sign in / Sign up

Export Citation Format

Share Document