Mg-nucleotides induced dissociation of liposome-bound creatine kinase: reversible changes in its secondary structure and in the fluidity of the bilayer

MgADP binding to mitochondrial creatine kinase (mtCK) adsorbed on liposomes was induced by the photorelease of caged ADP. The nucleotide binding produced two types of structural changes. One was related to the well-established release of mtCK from the liposomes. The other corresponded to reversible structural changes induced by nucleotide binding to mtCK as demonstrated here. Infrared spectroscopy data show that the MgADP-induced desorption of mtCK from vesicles led to a slight increase in &#102 -helix structures in mtCK at the expense of a small decrease in &#103 -sheet structures and a concomitant increase in the fluidity of the membranes. The desorption of mtCK induced by MgADP and MgATP was almost complete, as shown by centrifugation and enzymatic activity measurements. The photorelease of MgADP in a reactive medium containing phosphocreatine and mtCK associated with liposomes led to nucleotide binding and to the formation of MgATP and creatine. Addition of phosphocreatine also desorbed mtCK from liposomes, while addition of creatine did not. Interpretation of these results would suggest that ADP, ATP or phosphocreatine induce the release of mtCK from membranes, increase the phospholipid bilayer fluidity, and may also decrease the number of contact sites between inner and outer mitochondrial membranes, thus affecting the activity of other mitochondrial enzymes. It is tempting to propose that membrane mtCK binding regulation by nucleotide and PCr concentrations may serve as a physiological adaptation for energy supply.     

Analysis of functional coupling: mitochondrial creatine kinase and adenine nucleotide translocase

The mechanism of functional coupling between mitochondrial creatine kinase (MiCK) and adenine nucleotide translocase (ANT) in isolated heart mitochondria is analyzed. Two alternative mechanisms are studied: 1), dynamic compartmentation of ATP and ADP, which assumes the differences in concentrations of the substrates between intermembrane space and surrounding solution due to some diffusion restriction and 2), direct transfer of the substrates between MiCK and ANT. The mathematical models based on these possible mechanisms were composed and simulation results were compared with the available experimental data. The first model, based on a dynamic compartmentation mechanism, was not sufficient to reproduce the measured values of apparent dissociation constants of MiCK reaction coupled to oxidative phosphorylation. The second model, which assumes the direct transfer of substrates between MiCK and ANT, is shown to be in good agreement with experiments—i.e., the second model reproduced the measured constants and the estimated ADP flux, entering mitochondria after the MiCK reaction. This model is thermodynamically consistent, utilizing the free energy profiles of reactions. The analysis revealed the minimal changes in the free energy profile of the MiCK-ANT interaction required to reproduce the experimental data. A possible free energy profile of the coupled MiCK-ANT system is presented.     

The flavonoid quercetin induces changes in mitochondrial permeability by inhibiting adenine nucleotide translocase

This study shows the effects of the flavonoid quercetin on diverse mitochondrial functions, among them membrane permeability. Our findings indicate that the addition of 50 μM quercetin did not produce reactive oxygen derived species; however, it inhibited the oxidative stress induced after the addition of Fe²/H₂O₂ by about 38%. At this concentration, quercetin also promoted a fast calcium release, inhibited oxidative phosphorylation, stimulated oxygen consumption, and decreased membrane potential. In addition 50 μM quercetin inhibited the adenine nucleotide translocase (ANT) by 46%. These effects induced the opening of the permeability transition pore and release of cytochrome c, by its interaction with a component of the non-specific pore complex, fixed to the carrier in the conformation c, as carboxyatractyloside does. Quercetin-induced permeability transition pore opening was inhibited by 0.5 μM cyclosporin A, but, interestingly, the release of cytochrome c was not inhibited by the immunosuppressor, as quercetin was found to disrupt the outer membrane.     

Once again about the functional coupling between mitochondrial creatine kinase and adenine nucleotide translocase

The synthesis of creatine phosphate (CP) by mitochondrial creatine kinase during oxidative phosphorylation was terminated when the mass action ratio of the creatine kinase reaction = [ADP]·[CP][ATP]·[Cr] became equal to the apparent equilibrium constant (K _eq ^app) of this reaction. Subsequent excess of over the K _eq ^app was due to an increase in the ADP concentration in the medium. A comparable increase in the ADP concentration also occurred in the absence of creatine (Cr) in the incubation medium. Increase in the ADP concentration was shown to be associated with a decrease in the rate of oxidative phosphorylation and with a relative increase in the ATPase activity of mitochondria during the incubation. A low concentration of ADP (<30 M) and relatively high concentrations (1-6 mM) of other components of the creatine kinase reaction prevented the detection of the reverse reaction within 10 min after exceeded the K _eq ^app, but the reverse reaction became evident on more prolonged incubation. The reverse reaction was accompanied by a further increase in . Low ADP concentration in the medium was also responsible for the lack of an immediate conversion of the excess creatine phosphate added although > K _eq ^app. The findings are concluded to be in contradiction with the concept of microcompartment formation between mitochondrial creatine kinase and adenine nucleotide translocase.     

Inhibition of cytosolic and mitochondrial creatine kinase by siRNA in HaCaT- and HeLaS3-cells affects cell viability and mitochondrial morphology

The creatine kinase (CK) system is essential for cellular energetics in tissues or cells with high and fluctuating energy requirements. Creatine itself is known to protect cells from stress-induced injury. By using an siRNA approach to silence the CK isoenzymes in human keratinocyte HaCaT cells, expressing low levels of cytoplasmic CK and high levels of mitochondrial CK, as well as HeLa cancer cells, expressing high levels of cytoplasmic CK and low levels of mitochondrial CK, we successfully lowered the respective CK expression levels and studied the effects of either abolishing cytosolic brain-type BB-CK or ubiquitous mitochondrial uMi-CK in these cells. In both cell lines, targeting the dominant CK isoform by the respective siRNAs had the strongest effect on overall CK activity. However, irrespective of the expression level in both cell lines, inhibition of the mitochondrial CK isoform generally caused the strongest decline in cell viability and cell proliferation. These findings are congruent with electron microscopic data showing substantial alteration of mitochondrial morphology as well as mitochondrial membrane topology after targeting uMi-CK in both cell lines. Only for the rate of apoptosis, it was the least expressed CK present in each of the cell lines whose inhibition led to the highest proportion of apoptotic cells, i.e., downregulation of uMi-CK in case of HeLaS3 and BB-CK in case of HaCaT cells. We conclude from these data that a major phenotype is linked to reduction of mitochondrial CK alone or in combination with cytosolic CK, and that this effect is independent of the relative expression levels of Mi-CK in the cell type considered. The mitochondrial CK isoform appears to play the most crucial role in maintaining cell viability by stabilizing contact sites between inner and outer mitochondrial membranes and maintaining local metabolite channeling, thus avoiding transition pore opening which eventually results in activation of caspase cell-death pathways.     

Ca binding to c-state of adenine nucleotide translocase (ANT)-surrounding cardiolipins enhances (ANT)-Cys relative mobility: A computational-based mitochondrial permeability transition study

The oxidation of critical cysteines/related thiols of adenine nucleotide translocase (ANT) is believed to be an important event of the Ca²⁺-induced mitochondrial permeability transition (MPT), a process mediated by a cyclosporine A/ADP-sensitive permeability transition pores (PTP) opening. We addressed the ANT-Cys⁵⁶ relative mobility status resulting from the interaction of ANT/surrounding cardiolipins with Ca²⁺ and/or ADP by means of computational chemistry analysis (Molecular Interaction Fields and Molecular Dynamics studies), supported by classic mitochondrial swelling assays. The following events were predicted: (i) Ca²⁺ interacts preferentially with the ANT surrounding cardiolipins bound to the H4 helix of translocase, (ii) weakens the cardiolipins/ANT interactions and (iii) destabilizes the initial ANT-Cys⁵⁶ residue increasing its relative mobility. The binding of ADP that stabilizes the conformation “m” of ANT and/or cardiolipin, respectively to H5 and H4 helices, could stabilize their contacts with the short helix h56 that includes Cys⁵⁶, accounting for reducing its relative mobility. The results suggest that Ca²⁺ binding to adenine nucleotide translocase (ANT)-surrounding cardiolipins in c-state of the translocase enhances (ANT)-Cys⁵⁶ relative mobility and that this may constitute a potential critical step of Ca²⁺-induced PTP opening.     

Acidic lipids, H-ATPases, and mechanism of oxidative phosphorylation. Physico-chemical ideas 30 years after P. Mitchell's Nobel Prize award

Peter D. Mitchell, who was awarded the Nobel Prize in Chemistry 30 years ago, in 1978, formulated the chemiosmotic theory of oxidative phosphorylation. This review initially analyzes the major aspects of this theory, its unresolved problems, and its modifications. A new physico-chemical mechanism of energy transformation and coupling of oxidation and phosphorylation is then suggested based on recent concepts regarding proteins, including ATPases that work as molecular motors, and acidic lipids that act as hydrogen ion (H⁺) carriers. According to this proposed mechanism, the chemical energy of a redox substrate is transformed into nonequilibrium states of electron-transporting chain (ETC) coupling proteins. This leads to nonequilibrium pumping of H⁺ into the membrane. An acidic lipid, cardiolipin, binds with this H⁺ and carries it to the ATP-synthase along the membrane surface. This transport generates gradients of surface tension or electric field along the membrane surface. Hydrodynamic effects on a nanolevel lead to rotation of ATP-synthase and finally to the release of ATP into aqueous solution. This model also explains the generation of a transmembrane protonmotive force that is used for regulation of transmembrane transport, but is not necessary for the coupling of electron transport and ATP synthesis.     

Crystal structure of mitochondrial fission complex reveals scaffolding function for mitochondrial division 1 (Mdv1) coiled coil

The mitochondrial fission machinery is best understood in the yeast Saccharomyces cerevisiae, where Fis1, Mdv1, and Dnm1 are essential components. Fis1 is a mitochondrial outer membrane protein that recruits the dynamin-related GTPase Dnm1 during the fission process. This recruitment occurs via Mdv1, which binds both Fis1 and Dnm1 and therefore functions as a molecular adaptor linking the two molecules. Mdv1 has a modular structure, consisting of an N-terminal extension that binds Fis1, a central coiled coil for dimerization, and a C-terminal WD40 repeat region that binds Dnm1. We have solved the crystal structure of a dimeric Mdv1-Fis1 complex that contains both the N-terminal extension and coiled-coil regions of Mdv1. Consistent with previous studies, Mdv1 binds Fis1 through a U-shaped helix-loop-helix motif, and dimerization of the Mdv1-Fis1 complex is mediated by the antiparallel coiled coil of Mdv1. However, the complex is surprisingly compact and rigid due to two additional contacts mediated by the surface of the Mdv1 coiled coil. The coiled coil packs against both Fis1 and the second helix of the Mdv1 helix-loop-helix motif. Mutational analyses showed that these contacts are important for mitochondrial fission activity. These results indicate that, in addition to dimerization, the unusually long Mdv1 coiled coil serves a scaffolding function to stabilize the Mdv1-Fis1 complex.     

Modification of liver mitochondrial lipids and of adenine nucleotide translocase and oxidative phosphorylation by cold adaptation

The decrease in respiration rate following thyroidectomy is preceded by changes in the lipid composition of the mitochondrial membrane (Hoch, F.L., Subramanian, C., Dhopeshwarkar, G.A. and Mead, J.F. (1981) Lipids 16, 328–334) and in concert, changes in the kinetic parameters of the adenine nucleotide translocase (Mak, I.T., Shrago, E. and Elson, C.E. (1981) Fed. Proc. 40, 398). To demonstrate that physiological adaptation also involves this sequence of events, rats were housed at 8°C for 3–4 weeks. Cold adaptation resulted in a modest (5%) increase in the unsaturation index for the mitochondrial fatty acids comprised of a significant increase in arachidonic acid and a reciprocal decrease in linoleic acid. Phospholipid analysis indicated that cold adaptation increased the mitochondrial phosphatidylethanolamine and reciprocally decreased the phosphatidylcholine content. Concomitantly, cold adaptation resulted in 25–30% increases in rat liver mitochondrial respiratory activities without changing the respiratory control or ADP/O ratios. The kinetic parameters of the adenine nucleotide translocase were determined by the back-exchange method (Pfaff, E. and Klingenberg, M. (1968) Eur. J. Biochem. 6, 66–79). At 0–4 and 10°C, the V_max and K_m of the cold-adapted rat liver adenine nucleotide translocase were not distinguishable from the control values. The K_i values determined by Dixon plot studies for atractylate and palmitoyl-CoA were also comparable between the two groups. However, at 25 and 37°C, cold-adapted rat liver adenine nucleotide translocase exhibited a 20% increase in V_max and a 20% decrease in K_m for external ADP. The results suggest that one adaption to a cold environment involves hormone-mediated changes in the lipid composition in the mitochondrial membranes which in turn modulate the adenine nucleotide translocase and subsequent respiratory activities.     

Functional coupling of adenine nucleotide translocase and mitochondrial creatine kinase is enhanced after exercise training in lung transplant skeletal muscle

Mechanisms responsible for limitation of exercise capacity in lung transplant recipients (LR) and benefits gained by exercise training were studied. Mitochondrial respiration parameters, energy transfer, and cell structure were assessed in vastus lateralis biopsies using the permeabilized fiber technique with histochemical and morphometric measurements. Twelve male controls (C) and 12 LR performed exercise training over 12 wk. Before exercise training, there were strong correlations between exercise capacity (maximal O(2) consumption and endurance time at 70% maximal power output) and cellular events, as assessed by percentage of type I fibers and apparent K(m) for exogenous ADP. Anticalcineurins were not involved in LR exercise limitation, since there were no differences in maximal mitochondrial rate of respiration before exercise training and no abnormalities in respiratory chain complexes compared with C. Training resulted in a significant increase in physiological parameters both at the cellular (apparent K(m) for exogenous ADP and stimulating effect of creatine) and integrated (maximal O(2) consumption, power output at ventilatory threshold, maximal power output, and endurance time at 70% maximal power output) levels in LR and C. After the training period, improvements in maximal O(2) consumption and in maximal mitochondrial rate of respiration were noted, as well as changes in endurance time and percentage of type I fibers. Because there were no changes in diameters and fiber types, baseline alteration of apparent K(m) for exogenous ADP and its improvement after training might be related to changes within the intracellular energetic units. After the training period, intracellular energetic units exhibited a higher control of mitochondrial respiration by creatine linked to a more efficient functional coupling adenine nucleotide translocase-mitochondrial creatine kinase, resulting in better exercise performances in C and LR.     

Setting up and running molecular dynamics simulations of membrane proteins

Molecular dynamics simulations have become a popular and powerful technique to study lipids and membrane proteins. We present some general questions and issues that should be considered prior to embarking on molecular dynamics simulation studies of membrane proteins and review common simulation methods. We suggest a practical approach to setting up and running simulations of membrane proteins, and introduce two new (related) methods to embed a protein in a lipid bilayer. Both methods rely on placing lipids and the protein(s) on a widely spaced grid and then 'shrinking' the grid until the bilayer with the protein has the desired density, with lipids neatly packed around the protein. When starting from a grid based on a single lipid structure, or several potentially different lipid structures (method 1), the bilayer will start well-packed but requires more equilibration. When starting from a pre-equilibrated bilayer, either pure or mixed, most of the structure of the bilayer stays intact, reducing equilibration time (method 2). The main advantages of these methods are that they minimize equilibration time and can be almost completely automated, nearly eliminating one time consuming step in MD simulations of membrane proteins.     

Coarse-grained molecular dynamics simulations of membrane proteins and peptides

Molecular dynamics (MD) simulations provide a valuable approach to the dynamics, structure, and stability of membrane-protein systems. Coarse-grained (CG) models, in which small groups of atoms are treated as single particles, enable extended (>100 ns) timescales to be addressed. In this study, we explore how CG-MD methods that have been developed for detergents and lipids may be extended to membrane proteins. In particular, CG-MD simulations of a number of membrane peptides and proteins are used to characterize their interactions with lipid bilayers. CG-MD is used to simulate the insertion of synthetic model membrane peptides (WALPs and LS3) into a lipid (PC) bilayer. WALP peptides insert in a transmembrane orientation, whilst the LS3 peptide adopts an interfacial location, both in agreement with experimental biophysical data. This approach is extended to a transmembrane fragment of the Vpu protein from HIV-1, and to the coat protein from fd phage. Again, simulated protein/membrane interactions are in good agreement with solid state NMR data for these proteins. CG-MD has also been applied to an M3-M4 fragment from the CFTR protein. Simulations of CFTR M3-M4 in a detergent micelle reveal formation of an alpha-helical hairpin, consistent with a variety of biophysical data. In an I231D mutant, the M3-M4 hairpin is additionally stabilized via an inter-helix Q207/D231 interaction. Finally, CG-MD simulations are extended to a more complex membrane protein, the bacterial sugar transporter LacY. Comparison of a 200 ns CG-MD simulation of LacY in a DPPC bilayer with a 50 ns atomistic simulation of the same protein in a DMPC bilayer shows that the two methods yield comparable predictions of lipid-protein interactions. Taken together, these results demonstrate the utility of CG-MD simulations for studies of membrane/protein interactions.     

The transmembrane prolines of the mitochondrial ADP/ATP carrier are involved in nucleotide binding and transport and its biogenesis

The mitochondrial ADP/ATP carrier (Ancp) is a paradigm of the mitochondrial carrier family, which allows cross-talk between mitochondria, where cell energy is mainly produced, and cytosol, where cell energy is mainly consumed. The members of this family share numerous structural and functional characteristics. Resolution of the atomic structure of the bovine Ancp, in a complex with one of its specific inhibitors, revealed interesting features and suggested the involvement of some particular residues in the movements of the protein to perform translocation of nucleotides from one side of the membrane to the other. They correspond to three prolines located in the odd-numbered transmembrane helices (TMH), Pro-27, Pro-132, and Pro-229. The corresponding residues of the yeast Ancp (Pro-43, Ser-147, and Pro-247) were mutated into alanine or leucine, one at a time and analysis of the various mutants evidenced a crucial role of Pro-43 and Pro-247 during nucleotide transport. Beside, replacement of Ser-147 with proline does not inactivate Ancp and this is discussed in view of the conservation of the three prolines at equivalent positions in the Ancp sequences. These prolines belong to the signature sequences of the mitochondrial carriers and we propose they play a dual role in the mitochondrial ADP/ATP carrier function and biogenesis. Unexpectedly their mutations cause more general effects on mitochondrial biogenesis and morphology, as evidenced by measurements of respiratory rates, cytochrome contents, and also clearly highlighted by fluorescence microscopy.     

Molecular dynamics simulations of bovine rhodopsin: influence of protonation states and different membrane-mimicking environments

G-protein coupled receptors (GPCRs) are a protein family of outstanding pharmaceutical interest. GPCR homology models, based on the crystal structure of bovine rhodopsin, have been shown to be valuable tools in the drug-design process. The initial model is often refined by molecular dynamics (MD) simulations, a procedure that has been recently discussed controversially. We therefore analyzed MD simulations of bovine rhodopsin in order to identify contacts that could serve as constraints in the simulation of homology models. Additionally, the effect of an N-terminal truncation, the nature of the membrane mimic, the influence of varying protonation states of buried residues and the importance of internal water molecules was analyzed. All simulations were carried out using the program-package GROMACS. While N-terminal truncation negatively influenced the overall protein stability, a stable simulation was possible in both solvent environments. As regards the protonation state of titratable sites, the experimental data could be reproduced by the program UHBD (University of Houston Brownian Dynamics), suggesting its application for studying homology models of GPCRs. A high flexibility was observed for internal water molecules at some sites. Finally, interhelical hydrogen-bonding interactions could be derived, which can now serve as constraints in the simulations of GPCR homology models.     

Effects of deformability and thermal motion of lipid membrane on electroporation: by molecular dynamics simulations

Effects of mechanical properties and thermal motion of POPE lipid membrane on electroporation were studied by molecular dynamics simulations. Among simulations in which specific atoms of lipids were artificially constrained at their equilibrium positions using a spring with force constant of 2.0 kcal/(mol Ų) in the external electric field of 1.4 kcal/(mol Å e), only constraint on lateral motions of lipid tails prohibited electroporation while non-tail parts had little effects. When force constant decreased to 0.2 kcal/(mol Ų) in the position constraints on lipid tails in the external electric field of 2.0 kcal/(mol Å e), water molecules began to enter the membrane. Position constraints of lipid tails allow water to penetrate from both sides of membrane. Thermal motion of lipids can induce initial defects in the hydrophobic core of membrane, which are favorable nucleation sites for electroporation. Simulations at different temperatures revealed that as the temperature increases, the time taken to the initial pore formation will decrease.     

Pleiotropic effects of the yeast Sal1 and Aac2 carriers on mitochondrial function via an activity distinct from adenine nucleotide transport

In Saccharomyces cerevisiae, SAL1 encodes a Ca2+ -binding mitochondrial carrier. Disruption of SAL1 is synthetically lethal with the loss of a specific function associated with the Aac2 isoform of the ATP/ADP translocase. This novel activity of Aac2 is defined as the V function (for Viability of aac2 sal1 double mutant), which is independent of the ATP/ADP exchange activity required for respiratory growth (the R function). We found that co-inactivation of SAL1 and AAC2 leads to defects in mitochondrial translation and mitochondrial DNA (mtDNA) maintenance. Additionally, sal1Delta exacerbates the respiratory deficiency and mtDNA instability of ggc1Delta, shy1Delta and mtg1Delta mutants, which are known to reduce mitochondrial protein synthesis or protein complex assembly. The V function is complemented by the human Short Ca2+ -binding Mitochondrial Carrier (SCaMC) protein, SCaMC-2, a putative ATP-Mg/Pi exchangers on the inner membrane. However, mitochondria lacking both Sal1p and Aac2p are not depleted of adenine nucleotides. The Aac2R252I and Aac2R253I variants mutated at the R252-254 triplet critical for nucleotide transport retain the V function. Likewise, Sal1p remains functionally active when the R479I and R481I mutations were introduced into the structurally equivalent R479-T480-R481 motif. Finally, we found that the naturally occurring V-R+ Aac1 isoform of adenine nucleotide translocase partially gains the V function at the expense of the R function by introducing the mutations P89L and A96 V. Thus, our data support the view that the V function is independent of adenine nucleotide transport associated with Sal1p and Aac2p and this evolutionarily conserved activity affects multiple processes in mitochondria.     

Skeletal muscle mitochondrial free-fatty-acid content and membrane potential sensitivity in different thyroid states: involvement of uncoupling protein-3 and adenine nucleotide translocase

The effect of triiodothyronine (T3) on mitochondrial efficiency could be related to an increase in the concentrations of some proteins, such as uncoupling proteins (UCPs). Free fatty acids (FFA) seem to be a cofactor essential for the uncoupling activity of UCP3. In this paper, we report that the hypothyroidism-hyperthyroidism transition is accompanied by increases: (i) in the endogenous levels of mitochondrial FFA and (ii) in the sensitivity to FFA shown by the mitochondrial respiration rate and membrane potential, which correlated with the level of UCP3 protein. The level of the mRNA for adenine-nucleotide translocase-1 (ANT) was not affected by the thyroid state, while the ANT contribution to FFA-induced changes in mitochondrial uncoupling was low in the hypothyroid and euthyroid states but became more relevant in the hyperthyroid state at the highest concentration of FFA.     

Molecular dynamics simulations of microstructure and mixing dynamics of cryoprotective solvents in water and in the presence of a lipid membrane

Molecular dynamics (MD) simulation is used to investigate the solubility behavior of cryoprotective (CP) solvents, such as DMSO, ethylene glycol (EG) and glycerol (GL), in pure water and in the presence of a lipid membrane. The MD study is focused on an equilibration timescale required for mixing large CP aggregates with aqueous and aqueous/lipid environments. The MD analysis demonstrates that DMSO mixes rapidly with water, so that all solute molecules are uniformly distributed in the equilibrium aqueous solution. Our investigation of the microstructure of binary EG/water and GL/water systems reveals that, despite the miscibility of both CP solvents with water, they are not ideally mixed in aqueous solutions at the molecular level. The MD simulations show that the mixing dynamics of the large CP cluster and surrounding water is found to be strongly dependent on nature of hydrophilic and hydrophobic interactions acting between cryoprotectant molecules. In particular, a spatial hydrogen-bond network formed between CP molecules plays an important role in the mixing dynamics between CP agents and water. A further analysis on the mixing behavior of the CP solvents with pure water and with aqueous solutions at a lipid membrane interface shows that, due to strong binding of the CP molecules to membrane surface, the equilibration process in the lipid environment becomes very slow, at least of the order of microseconds. The MD results are discussed in the context of the better understanding on the composition of the aqueous mixtures of the EG and GL solvents. Knowledge of the microstructure and the dynamics of these systems helps to develop better cryopreservation protocols and to propose more optimal cooling/warming regimes for cellular cryosolutions.     

Identification of Tom5 and Tom6 in the preprotein translocase complex of human mitochondrial outer membrane

The fungal preprotein translocase of the mitochondrial outer membrane (TOM complex) comprises import receptors Tom70, Tom20, and Tom22, import channel Tom40, and small Tom proteins Tom5, Tom6, and Tom7, which regulate TOM complex assembly. These components are conserved in mammals; unlike the other components, however, Tom5 and Tom6 remain unidentified in mammals. We immuno-isolated the TOM complex from HeLa cells expressing hTom22-FLAG and identified the human counterparts of Tom5 and Tom6, together with the other components including Tom7. These small Tom proteins are associated with Tom40 in the TOM complex. Knockdown of Tom7, but not Tom5 and Tom6, strongly compromised stability of the TOM complex. Conversely, knockdown of hTom40 decreased the level of all small Tom proteins. Matrix import of preprotein was affected by double knockdown of any combination of small Tom proteins. These results indicate that human small Tom proteins maintain the structural integrity of the TOM complex.     

Refolding molecular dynamics simulations of small- and middle-sized proteins in an explicit solvent

In order to elucidate the protein folding problem, we performed molecular dynamics simulations for small- and middle-sized two unfolding and six refolding proteins in an explicit solvent. Histidine-containing phosphocarrier protein and small designed protein were chosen for the simulations. We found that the protein folding process of these proteins was divided into three phases: an α -helix formation phase, a packing phase and a β -sheet formation phase. In the α -helix formation phase, an α -helix was developed from a β -turn structure through a 3₁₀-helix state. In the packing phase, proteins became compact, and tertiary structures (α / α or pre- β / β packing) were formed. Formation of a hydrophobic nucleus occurred concomitant with the α -helix formation and packing phase. Finally, in the β -sheet formation phase, a β -sheet was developed owing to the sequential formation of hydrogen bonds between two neighbouring strands, just like a "closing zipper".