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Mitochondrial Membrane, Inner

The inner mitochondrial membrane is a critical component of the mitochondrial structure, playing a vital role in cellular energy production.
This membrane is highly specialized, containing numerous protein complexes involved in the electron transport chain and oxidative phosphorylation process.
The inner membrane is also responsible for maintaining the proton gradient necessary for ATP synthesis.
Researchers studying the inner mitochondrial membrane can leverage PubCompare.ai's AI-driven protocol optimization platform to streamline their work and enhance reproducibility.
By accessing the best protocols from literature, preprints, and patents, scientists can idnetify the most accurate and reliable methods to advance their mitochondrial membrane research.

Most cited protocols related to «Mitochondrial Membrane, Inner»

1
Engineering Redox-Sensitive RoGFP Sensors
The RoGFP protein contains two engineered cysteine thiols, as first described by Remington et al. (RoGFP2) 11 (link). The cDNA encoding the protein was created by introducing four mutations in the mammalian GFP expression vector (pEGFP-N1) (C48S, Q80R, S147C, and Q204C) using a QuikChange Multi Site-directed mutagenesis kit (Strategene). The RoGFP construct was ligated into the VQ Ad5CMV K-NpA adenoviral shuttle vector between the KpnI and NotI sites; after sequencing and amplification this plasmid was used to generate a recombinant adenovirus to permit widespread expression in our cells (ViraQuest Inc., North Liberty, IA). The resulting redox-sensitive protein has excitation maxima at 400 and 484 nm, with emission at 525 nm. In response to changes in redox conditions, RoGFP exhibits reciprocal changes in intensity at the two excitation maxima 12 (link), and its ratiometric characteristics render it insensitive to expression levels 13 (link)-15 (link). Although RoGFP’s fluorescence behavior is relatively independent of pH and it does not respond to authentic nitric oxide (NO), reduced NADH, or the antioxidant N-acetyl-L-cysteine (NAC), its spectrum is slightly affected by reduced glutathione (GSH) possibly due to thiol-disulfide exchange (Online Figures I and II).
RoGFP was expressed in the mitochondrial matrix (Mito-RoGFP) by appending a 48 bp region encoding the mitochondrial targeting sequence from cytochrome oxidase subunit IV, at the 5′ end of the coding sequence. This construct was then ligated into the VQ Ad5CMV K-NpA plasmid between the KpnI and NotI sites, and used to generate an adenoviral vector. RoGFP was targeted to the mitochondrial inter-membrane space (IMS-RoGFP) by appending it to glycerol phosphate dehydrogenase (GPD). A cDNA construct encoding GPD, an integral protein of the inner mitochondrial membrane whose C-terminus protrudes into the inter-membrane space 17 (link), was ligated in-frame with cDNA encoding RoGFP 17 (link). The corresponding polypeptide includes amino acids 1–626 of GPD, with RoGFP at the carboxy terminus. This method has been used previously to express YFP in the inter-membrane space 18 (link). (See Online Supplemental Material for characterization of the RoGFP sensors and experimental protocols).
Waypa G.B., Marks J.D., Guzy R., Mungai P.T., Schriewer J., Dokic D, & Schumacker P.T. (2009). HYPOXIA TRIGGERS SUBCELLULAR COMPARTMENTAL REDOX SIGNALING IN VASCULAR SMOOTH MUSCLE CELLS. Circulation research, 106(3), 526-535.
Publication 2009
Acetylcysteine Adenoviruses Adenovirus Vaccine Amino Acids Antioxidants Cells Cloning Vectors Cysteine Cytochrome-c Oxidase Subunit IV Disulfides DNA, Complementary Fluorescence glycerol-1-phosphate dehydrogenase Glycerol-3-Phosphate Dehydrogenase Integral Membrane Proteins Mammals Mitochondria Mitochondrial Membrane, Inner Mitochondrial Membranes Mitomycin Mutagenesis, Site-Directed Mutation NADH Open Reading Frames Oxidation-Reduction Oxide, Nitric Plasmids Polypeptides Proteins Reading Frames Reduced Glutathione Shuttle Vectors Sulfhydryl Compounds Tissue, Membrane
2
Measuring Cellular Bioenergetics with Seahorse
Cellular bioenergetics of the isolated cells was determined using the extracellular flux analyzer (Seahorse Bioscience), which measures O2 and protons. This system allows for real-time, noninvasive measurements of O2 consumption rate (OCR) and proton production rate (PPR), which can be correlated to mitochondrial function/oxidative burst and glycolysis, respectively.43 (link) The injection ports attached to the wells allow for injection of inhibitors of mitochondrial respiratory chain or activators of the oxidative burst to determine the defects in individual cellular respiration pathways or enzymes. Pilot experiments for monocytes, neutrophils, platelets, and lymphocytes isolated from individual donors were performed to determine the optimal cell number required for accurate measurements of OCR and PPR. The optimum concentration of the inhibitors and activators to be used for the assessment of mitochondrial function and oxidative burst were determined by titrating the individual compounds in separate experiments against the cell number determined in the first set of experiments. First, the mean basal respiration is determined by taking 3–4 OCR measurements before the addition of the inhibitors or activators. ATP-linked OCR and proton leak were determined by injecting oligomycin at 0.5 μM (for monocytes, lymphocytes, and neutrophils) or 0.75 μM (for platelets). The fall in OCR following oligomycin injection is the rate of oxygen consumption that corresponds to ATP synthesis, and the oligomycin-insensitive rate is considered as proton leak across the inner mitochondrial membrane. FCCP, an uncoupler of the electron transport chain, was used at a concentration of 0.6 μM to determine the maximal respiration rate. This rate gives the theoretical maximum oxygen consumption that can take place at cytochrome c oxidase whether limited by availability of substrate or activity of the electron transport chain. The difference between the basal rate and this FCCP-stimulated rate is the reserve capacity of the mitochondrion, which is a measure of the maximal potential respiratory capacity the cell can utilize under conditions of stress and/or increased energetic demands. Antimycin A, an inhibitor of Complex III, was used to completely inhibit mitochondrial electron transport. The OCR determined after antimycin A injection is attributable to non-mitochondrial oxygen consumption. Mitochondrial basal respiration, proton leak, and the maximal respiration were calculated after correcting for the non-mitochondrial OCR for each assay. Cells were allowed to attach to the XF24 plate for 30–60 min before measurement of mitochondrial function. Under these conditions, viability was over 90% for all cell types and remained so over the time course of the assay. At the end of the assay period, cell lysates were collected, and OCR and PPR values normalized to the protein content in each well.
Chacko B.K., Kramer P.A., Ravi S., Johnson M.S., Hardy R.W., Ballinger S.W, & Darley-Usmar V.M. (2013). Methods for defining distinct bioenergetic profiles in platelets, lymphocytes, monocytes, and neutrophils, and the oxidative burst from human blood. Laboratory Investigation; a Journal of Technical Methods and Pathology, 93(6), 690-700.
Publication 2013
Anabolism Antimycin A Bioenergetics Biological Assay Biological Transport, Active Blood Platelets Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone Cardiac Arrest Cell Respiration Cells Donors Electrons Electron Transport Electron Transport Complex III Enzymes Glycolysis inhibitors Lymphocyte Mitochondria Mitochondrial Membrane, Inner Monocytes Neutrophil Oligomycins Oxidase, Cytochrome-c Oxygen Consumption Proteins Protons Respiratory Burst Respiratory Chain Respiratory Rate Seahorses Stress Disorders, Traumatic
3
Mitochondrial ion channel characterization
Patch-clamp experiments using mitoplasts were performed as described previously [18] (link), [19] (link). Briefly, mitoplasts were prepared from a sample of human astrocytoma mitochondria placed in a hypotonic solution (5 mM HEPES, 200 µM CaCl2, pH = 7.2) for approximately 1 min to induce swelling and breakage of the outer membrane. Then, a hypertonic solution (750 mM KCl, 30 mM HEPES, 200 µM CaCl2, pH = 7.2) was added to restore the isotonicity of the medium. The patch-clamp pipette was filled with an isotonic solution containing 150 mM KCl, 10 mM HEPES, and 200 µM CaCl2 at pH = 7.2. Mitoplasts are easily recognizable due to their size, round shape, transparency, and presence of a “cap”, characteristics that distinguish these structures from the cellular debris that is also present in the preparation. An isotonic solution containing 200 µM CaCl2 was used as the control solution for all of the presented data. The low-calcium solution (1 µM CaCl2) contained the following: 150 mM KCl, 10 mM HEPES, 1 mM EGTA and 0.752 mM CaCl2 at pH = 7.2. All of the modulators of the channels and the substrates and inhibitors of the respiratory chain were added as dilutions in isotonic solution containing 200 µM CaCl2. To apply these substances, we used a perfusion system containing a holder with a glass tube (made in our workshop), a peristaltic pump, and Teflon tubing. The mitoplasts at the tip of the measuring pipette were transferred into the openings of a multibarrel “sewer pipe” system in which their outer faces were rinsed with the test solutions (Fig. 1A). The configuration of our patch-clamp mode is presented in Fig. 1A. The experiments were carried out in patch-clamp inside-out mode. This is based on observations with various mitochondrial substrates applied such as NADH or succinate. Reported voltages are those applied to the patch-clamp pipette interior. Hence, positive potentials represent the physiological polarization of the inner mitochondrial membrane (outside positive).
The electrical connection was made using Ag/AgCl electrodes and an agar salt bridge (3 M KCl) as the ground electrode. The current was recorded using a patch-clamp amplifier (Axopatch 200B, Molecular Devices Corporation, USA). The pipettes, made of borosilicate glass, had a resistance of 10–20 MΩ and were pulled using a Flaming/Brown puller.
The currents were low-pass filtered at 1 kHz and sampled at a frequency of 100 kHz. The traces of the experiments were recorded in single-channel mode. The illustrated channel recordings are representative of the most frequently observed conductance for the given condition. The conductance of the channel was calculated from the current-voltage relationship (data not shown). The probability of channel opening (Po, open probability) was determined using the single-channel search mode of the Clampfit 10.2 software. Calculations were performed using segments of continuous recordings lasting 60 s, with N>1000 events. Data from the experiments are reported as the mean values ± standard deviations (S.D.). Student’s t-test was used for statistical analysis. In figures showing single-channel recordings, “-” indicates the closed state of the channel.
Bednarczyk P., Wieckowski M.R., Broszkiewicz M., Skowronek K., Siemen D, & Szewczyk A. (2013). Putative Structural and Functional Coupling of the Mitochondrial BKCa Channel to the Respiratory Chain. PLoS ONE, 8(6), e68125.
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Publication 2013
Agar Astrocytoma Calcium Cellular Structures Egtazic Acid Electricity Face HEPES Homo sapiens Hypertonic Solutions Hypotonic Solutions inhibitors Isotonic Solutions Medical Devices Mitochondria Mitochondrial Membrane, Inner NADH Perfusion Peristalsis physiology Respiratory Chain Sodium Chloride Student Succinate Technique, Dilution Teflon Tissue, Membrane
4
Mitochondrial Fractionation and Isolation
Cells from a 15-cm tissue culture dish were transfected and after the indicated treatments, were recovered by scraping in 1 ml PBS and centrifuged at 300× g for 5 min at 4 °C. The pellet was resuspended in KCl Respiration Buffer [140 mM KCl, 10 mM MgCl2, 10 mM 3-(N-morpholino)propanesulfonic acid, 5 mM KH2PO4, 1 mM ethylene glycol tetraacetic acid, 0.2% bovine serum albumin (fatty acid free; Sigma, A6003-25G)], supplemented with protease inhibitor cocktail. Cells were disrupted via Dounce homogenization and centrifuged at 600× g for 5min. The resulting supernatant fraction was centrifuged at 8000× g for 15 min to pellet the mitochondria and washed twice for 10 min at 8000× g. To swell the mitochondria in order to rupture the outer membrane, the mitochondrial pellet fraction was resuspended in hypotonic buffer (1 mM ethylene glycol tetraacetic acid, 10 mM potassium phosphate, pH 7.4) by trituration and stored on ice for 15 min; MgCl2 was supplemented to a final concentration of 1 mM for an additional 5 min, then the mitochondria were centrifuged at 16,000× g for 15 min at 4 °C. The resulting pellet fraction, designated the mitoplast fraction (mitochondrial inner membrane and matrix), was reserved for further processing, while the supernatant fraction, representing proteins from the outer mitochondrial membrane and the intermembrane space was centrifuged at 100,000× g for 60 min at 4 °C to obtain a pellet fraction of outer membrane proteins and a supernatant fraction containing intermembrane space constituents. The mitoplasts were resuspended in Respiration Buffer without albumin, sonicated, and centrifuged at 100,000× g for 60 min at 4 °C, to obtain a pellet fraction of inner membrane proteins and a supernatant fraction of matrix constituents. Pellet fractions were resuspended in consistent volumes to maintain mitochondria-equivalent fractions.
Hernandez G., Thornton C., Stotland A., Lui D., Sin J., Ramil J., Magee N., Andres A., Quarato G., Carreira R.S., Sayen M.R., Wolkowicz R, & Gottlieb R.A. (2013). MitoTimer: A novel tool for monitoring mitochondrial turnover. Autophagy, 9(11), 1852-1861.
Publication 2013
Acids Albumins Buffers Cell Culture Techniques Cell Respiration Cells Egtazic Acid Fatty Acids Hyperostosis, Diffuse Idiopathic Skeletal Integral Membrane Proteins Magnesium Chloride Mitochondria Mitochondrial Membrane, Inner Mitochondrial Membrane, Outer Morpholinos OMPA outer membrane proteins potassium phosphate Protease Inhibitors Proteins Serum Albumin, Bovine Tissue, Membrane Tissues
5
Mapping Mouse Gene Ontology Annotations
Mouse GO-BP annotations were downloaded from the Gene Ontology website [46 ] and the European Bioinformatics Institute (EBI) [47 ] and both were mapped to XM gene sequences by sequence identity to the annotated source sequences. The full annotation database is on our website [16 ]. Fewer than 0.01% of these annotations were derived from gene expression (IEP code); we confirmed that removal of these genes had no appreciable impact on statistical analysis or the SVM analysis, and hence the use of these annotations to analyze gene expression is not circular. The Mouse Genome Informatics (MGI) annotations are reported to be manually compiled, whereas the EBI annotations include automated sequence-based annotations (for example, potassium channels are annotated as being in 'ion transport' and the mouse homolog of the yeast Tim8 protein, which is a translocase of the inner mitochondrial membrane, is annotated as being in 'mitochondrial translocation'). All GO-BP annotations were propagated up all possible edges of the GO graph. Redundant GO-BP categories were excluded. Categories with fewer than three genes among the 21,622 expressed genes were excluded from our analysis since they are not appropriate for the statistical tests we used, and those with more than 500 genes were excluded because they are not specific to distinct physiological processes.
Zhang W., Morris Q.D., Chang R., Shai O., Bakowski M.A., Mitsakakis N., Mohammad N., Robinson M.D., Zirngibl R., Somogyi E., Laurin N., Eftekharpour E., Sat E., Grigull J., Pan Q., Peng W.T., Krogan N., Greenblatt J., Fehlings M., van der Kooy D., Aubin J., Bruneau B.G., Rossant J., Blencowe B.J., Frey B.J, & Hughes T.R. (2004). The functional landscape of mouse gene expression. Journal of Biology, 3(5), 21.
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Publication 2004
Europeans Gene Expression Genes Genome Ion Transport Mice, Laboratory Mitochondrial Inheritance Mitochondrial Membrane, Inner Physiological Processes Potassium Channel Translocation, Chromosomal Yeast Proteins

Most recents protocols related to «Mitochondrial Membrane, Inner»

1
Mitochondrial ATP Synthase Complex Analysis
Two-dimensional gel electrophoresis was based on the protocol of Schamel43 (link) with slight modifications. Briefly, the ATP synthase complexes were liberated from inner mitochondrial membrane of isolated mitochondria by incubation with 1–2% digitonin in extraction buffer (30 mM HEPES, 150 mM potassium acetate, 12% glycerol, 2 mM 6-aminocaproic acid, 1 mM EGTA, protease inhibitor cocktail tablets EDTA-free (Roche), pH 7.4) for different time intervals up to 60 min and separated using NativePAGE™ 3–12% Bis–Tris Gels (Thermo Fisher Scientific) to separate monomeric and dimeric ATP synthase complexes44 (link). For second dimensional analysis the lanes were cut from the gel and placed in SDS-PAGE running buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS, pH 8.3 with 1% β-mercaptoethanol), heated in a microwave for 10 secs and incubated for another 10 min in a shaker. The gel strips were then loaded on the top of a 16% SDS-PAGE gel, and electrophoresis was conducted under denaturing conditions. Then the gel was stained with Coomassie blue or silver staining and bands cut-off were analyzed by mass spectrometry. For Western blotting proteins from the gel were transferred into PVDF or nitrocellulose membranes using iBlot system (Thermo Fisher Scientific). For SDS-PAGE analysis of steady state level of proteins, yeast cells were disrupted by alkaline lysis with NaOH/TCA45 (link). Western blot analysis was performed using the polyclonal rabbit anti-Mco10 antibody, anti-ATP synthase subunits antibodies (gifts from Marie-France Giraud, Bordeaux, France and Martin van der Laan, Germany), anti-Rip1 and Cob1 antibodies (provided by dr hab. Ulrike Topf, IBB PAS) or anti-Cox2 (Thermo Fisher Scientific).
Panja C., Wiesyk A., Niedźwiecka K., Baranowska E, & Kucharczyk R. (2023). ATP synthase interactome analysis identifies a new subunit l as a modulator of permeability transition pore in yeast. Scientific Reports, 13, 3839.
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Publication 2023
2-Mercaptoethanol 6-Aminocaproic Acid Anti-Antibodies Antibodies Antibodies, Anti-Idiotypic Bistris Buffers Cells Coomassie blue Digitonin Edetic Acid Egtazic Acid Electrophoresis Electrophoresis, Gel, Two-Dimensional Gifts Glycerin Glycine HEPES Mass Spectrometry Microwaves Mitochondria Mitochondrial Membrane, Inner Nitric Oxide Synthase Nitrocellulose polyvinylidene fluoride Potassium Acetate Protease Inhibitors Proteins Protein Subunits PTGS2 protein, human Rabbits Saccharomyces cerevisiae SDS-PAGE Tissue, Membrane Tromethamine Western Blot
2
Measuring Mitochondrial Respiration in bEnd.3 Cells
As previously described, the high-resolution Oroboros Oxygraph 2K (Oroboros Instruments, Innsbruck, Austria) was used to assess mitochondrial respiration rates in bEnd.3 cells using two 2-ml chambers under continuous stirring at 37°C (Abdel-Rahman et al., 2016 (link); Roy Chowdhury et al., 2020 (link)). The treated bEnd.3 cells were washed with PBS and then suspended in DMEM medium and transferred into the O2k chambers. At the beginning of the experiment, the oxygen flux is monitored for at least 10 min to determine routine respiration. Oligomycin (Omy) is injected through the stoppers to detect LEAK respiration, which indicates that ATP synthase is inhibited and protons leak across the inner mitochondrial membrane. Next, Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) is sequentially injected into the chambers until the respiratory flux reaches a plateau to determine the maximum capacity of the electronic transmission system. The difference in values between maximum respiration and Routine respiration represents the spare respiratory capacity for mitochondrial ATP production (Roy Chowdhury et al., 2020 (link)). The respiratory control ratio is defined as the ratio of uncoupled respiration rates to the LEAK respiratory rates, and it represents the mitochondrial respiration capacity (Hall et al., 2013 (link); Roy Chowdhury et al., 2020 (link)). Data acquisition and graphic presentation were performed with the DatLab® software, version 4.3 (Oroboros Instruments).
Jia J., Deng J., Jin H., Yang J., Nan D., Yu Z., Yu W., Shen Z., Lu Y., Liu R., Wang Z., Qu X., Qiu D., Yang Z, & Huang Y. (2023). Effect of Dl-3-n-butylphthalide on mitochondrial Cox7c in models of cerebral ischemia/reperfusion injury. Frontiers in Pharmacology, 14, 1084564.
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Publication 2023
Carbonyl Cyanide m-Chlorophenyl Hydrazone Cell Respiration Cells Decompression Sickness Mitochondrial Inheritance Mitochondrial Membrane, Inner Nitric Oxide Synthase Oligomycins Oxygen Protons Respiratory Rate Transmission, Communicable Disease
3
DHODH Activity Assay for P. oryzae and H. sapiens Enzymes
Preparation of histidine-tagged recombinant P. oryzae DHODH (PoDHODH) and N-terminal truncated H. sapience DHODH (ΔN-HsDHODH) protein, that the N-terminal 1-28 amino acids truncated from full length HsDHODH takes a role of import and proper location and fixation of the enzyme in the inner mitochondrial membrane,9 (link)) is described in the supplementary material. The DHODH activity was measured using PoDHODH and ΔN-HsDHODH proteins following the previously described protocol.2 (link),8 (link),10 (link)) The oxidation of the substrate dihydroorotate with the quinone co-substrate was coupled to reduce the chromogen 2,6-dichloroindophenol (DCIP). One hundred microliters of reaction mixture containing 50-mM Tris-HCl (pH 8.0), 150-mM NaCl, 0.1% (w/v) TritonX-100, 200-μM DCIP, 2-mM dihydroorotate, 100-μM decylubiquinone (QD), approximately 10-µg/mL recombinant PoDHODH or ΔN-HsDHODH protein suspension, and various concentrations of test compounds dissolved in 1% DMSO (or no compound control) were incubated at 30°C for 20–30 min. After incubation, 10-µL 10% sodium dodecyl sulfate was added to each sample and mixed well to stop the reaction. Then, absorbance at 595-nm was measured. The inhibitory rate was calculated as (1-T/C), where C and T represent the decreased absorbance quantity at 595-nm with the control and test samples, respectively. The IC50 (half-inhibition concentration) values for the test compounds on PoDHODH and ΔN-HsDHODH were determined using a four-parameter logistic curve-fitting program (GraphPad Prism 6.00), in which two parameters were constrained (i.e., the top and bottom were fixed as 1 and 0).
Higashimura N., Hamada A, & Banba S. (2023). Novel fungicide quinofumelin shows selectivity for fungal dihydroorotate dehydrogenase over the corresponding human enzyme. Journal of Pesticide Science, 48(1), 17-21.
Publication 2023
2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone Amino Acids azo rubin S dihydroorotate Dihydroorotate Dehydrogenase Enzymes Histidine Mitochondrial Membrane, Inner prisma Proteins Psychological Inhibition Quinones Sodium Chloride Sulfate, Sodium Dodecyl Sulfoxide, Dimethyl Tromethamine
4
Mitochondrial Bioenergetics Profiling
To assess mitochondrial function, a Mito stress test on a Seahorse XFp analyzer (Agilent, Santa Clara, CA, USA) was performed. The entire culture media was replaced with Seahorse XFp Base medium (pH 7.4). Following the manufacturer’s protocol, the mitochondrial inhibitors oligomycin, FCCP (trifluoromethoxy carbonyl cyanide phenylhydrazone), and rotenone/antimycin A were consecutively injected into the ports of the cartridge. Injection of the first chemical, known as an ATP synthase inhibitor, determines the production of ATP. FCCP makes the inner mitochondrial membrane permeable to protons and allows maximum electron flux through the electron transport chain (ETC). Thus, collapse of the inner membrane gradient is induced, driving mitochondria to respire at the maximum rate. The latter of the chemicals directly inhibits Complex I of the ETC and shuts mitochondrial respiration down (Figure 1). Such a combination of inhibitors allows for the assessment of several parameters: basal and maximal respiration, ATP levels, and spare respiratory capacity, as well as other mitochondrial characteristics such as proton leak, and non-mitochondrial respiration (Figure 2). For each experiment, the oxygen consumption rate (OCR) was measured, and the data were analyzed using Seahorse Wave Desktop Software v2.6.0 (Agilent, Santa Clara, CA, USA).
Zahariev N., Draganova M., Zagorchev P, & Pilicheva B. (2023). Casein-Based Nanoparticles: A Potential Tool for the Delivery of Daunorubicin in Acute Lymphocytic Leukemia. Pharmaceutics, 15(2), 471.
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Publication 2023
Antimycin A Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone carbonyl cyanide phenylhydrazone Cardiac Arrest Cell Respiration Electron Transport Electron Transport Complex I Exercise Tests inhibitors Mitochondria Mitochondrial Membrane, Inner Mitomycin Nitric Oxide Synthase Oligomycins Oxygen Consumption Permeability Protons Respiratory Rate Rotenone Seahorses Shock Tissue, Membrane
5
Molecular Dynamics of SLC25A20 in IMM
SLC25A20 structural models have been inserted in a bilayer phospholipid membrane mimicking the inner mitochondrial membrane (IMM), using the web server CHARMM-GUI (http://www.charmm-gui.org, accessed on 1 December 2022) [62 (link)]. The membrane composition was based on the IMM model published by the CHARMM-GUI team available at the CHARMM-GUI Archive (https://charmm-gui.org/?doc=archive&lib=biomembrane, accessed on 1 December 2022) [63 (link)]. Different concentrations and lipid tail composition are used to better represent the inner and outer IMM leaflets. In this model membrane, phosphatidylcholine is the most represented phospholipid species, followed by phosphatidylethanolamine and cardiolipin, the latter being more abundant in the inner layer. Water molecules, from the TIP3P model, were added on both sides of the membrane, forming two layers each 22.5 Å thick. The total system charge was neutralized by adding NaCl ions, reaching a physiological concentration of 0.15 M. The CHARMM36m force field [64 (link)] and the AMBER22 package [65 ] were used to perform the MD simulations of the assembled systems (~100,000 atoms, see Supplementary Table S6 for representative compositions), following the CHARMM-GUI protocol. First, an energy minimization procedure, involving 2500 steps of steepest descent and 2500 steps of conjugate gradient, was performed. Positional restraints were applied on the protein residues (10 kcal mol−1 Å−2) and on the membrane (2.5 kcal mol−1 Å−2). The resulting minimized systems have then been simulated using the canonical NVT ensemble, reaching a final temperature of 310.15 K. Thereafter, isothermal-isobaric NPT ensemble simulations have been performed to equilibrate the pressure to 1 bar. During the thermalization and equilibration phases, the positional restraints have been gradually reduced. The equilibrated system, without restraints, was properly simulated using the NPT ensemble for a total of 1 μs. Each system was simulated in replica. The Langevin thermostat has been used for both NVT and NPT ensembles, while the Monte Carlo barostat with a semiisotropic pressure scaling has been used for pressure control [66 (link)]. All of the MD simulations have been performed in a periodic boundary system. For the long range non-bonded interactions, the Particle Mesh Ewald method [67 (link)] and a 12 Å cut-off, with a force switching region at 10 Å, were used. The MD simulations were performed with a time step of 2 fs, apart from the apoSLC25A20 simulation for which the hydrogen mass repartitioning (HMR) method [68 (link)] was used with a 4 fs time step.
Pasquadibisceglie A., Quadrotta V, & Polticelli F. (2023). In Silico Analysis of the Structural Dynamics and Substrate Recognition Determinants of the Human Mitochondrial Carnitine/Acylcarnitine SLC25A20 Transporter. International Journal of Molecular Sciences, 24(4), 3946.
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Publication 2023
Cardiolipins Hydrogen Ions Lipids Mitochondrial Membrane, Inner Mitochondrial Membrane, Outer Phosphatidylcholines Phosphatidylethanolamines Phospholipids physiology Pressure Proteins Sodium Chloride STEEP1 protein, human Tail Tissue, Membrane

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More about "Mitochondrial Membrane, Inner"

The inner mitochondrial membrane is a crucial component of the mitochondrial structure, playing a vital role in cellular energy production.
This highly specialized membrane contains numerous protein complexes involved in the electron transport chain and oxidative phosphorylation process.
It is responsible for maintaining the proton gradient necessary for ATP synthesis.
Researchers studying the inner mitochondrial membrane can leverage advanced tools and techniques to streamline their work and enhance reproducibility.
Oxygraph-2k and XFp Extracellular Flux Analyzer are powerful instruments that can be used to measure oxygen consumption rates and mitochondrial function within cells.
The Infinite M200 Pro and XF24 Extracellular Flux Analyzer are other options for real-time analysis of mitochondrial respiration.
Fluorescent probes like the JC-1 dye and NIS-Elements AR 3.10 software can be employed to visualize and quantify mitochondrial membrane potential. 24-well plates and SYBR Green are common tools used in experimental setups.
The NIR Mitochondrial Membrane Potential Assay kit and MitoTracker Green FM provide alternative methods for assessing mitochondrial health and activity.
By accessing the best protocols from literature, preprints, and patents, scientists can identify the most accurate and reliable methods to advance their mitochondrial membrane research.
PubCompare.ai's AI-driven protocol optimization platform can be leveraged to streamline this process and enhance reproducibility.
This innovative solution helps researchers locate and compare the most effective procedures, boosting the efficiency and impact of their mitochondrial membrane studies.