Structural Basis of DEPTOR to Recognize Phosphatidic Acid Using its Tandem DEP Domains

DEP domain containing mTOR-interacting protein (DEPTOR) plays pivotal roles in regulating metabolism, growth, autophagy and apoptosis by functions as an endogenous inhibitor of mTOR signaling pathway. Activated by phosphatidic acid, a second messenger in mTOR signaling, DEPTOR dissociates from mTORC1 complex with unknown mechanism. Here, we present a 1.5 Å resolution crystal structure, which shows that the N-terminal two tandem DEP domains of hDEPTOR fold into a dumbbell-shaped structure, protruding the characteristic β-hairpin arms of DEP domains on each side. An 18 amino acids DDEX motif at the end of DEP2 interacts with DEP1 and stabilizes the structure. Biochemical studies showed that the tandem DEP domains directly interact with phosphatidic acid using two distinct positively charged patches. These results provide insights into mTOR activation upon phosphatidic acid stimulation.     

Dimerization of Cadherin-11 involves multi-site coupled unfolding and strand swapping

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Biofilter performance and characterization of a biocatalyst degrading alkylbenzene gases

A biofilter treating alkylbenzene vapors was characterized for its optimal running conditions and kinetic parame-ters. Kinetics of the continuous biofilter were compared to batch kinetic data obtained with biofilm samples as well as with defined microbial consortia and with pure culture isolates from the biofilter. Both bacteria and fungi were present in the bioreactor. Five strains were isolated. Two bacteria, Bacillus and Pseudomonas, were shown to be dominant, as well as a Trichosporon strain which could, however, hardly grow on alkylbenzenes in pure culture. The remaining two strains were most often overgrown by the other three organisms in liquid phase batch cultures μ _max, K_S, K_I values and biodegradation rates were calculated and compared for the difterent mixed and pure cultures. Since filter bed acidification was observed during biofiltration studies reaching a pH of about 4, experiments were also undertaken to study the influence of pH on performance of the different cultures. Biodegradation and growth were possible in all cases, over the pH range 3.5–7.0 at appreciable rates, both with mixed cultures and with pure bacterial cultures. Under certain conditions, microbial activity was even observed in the presence of alkylbenzenes down to pH 2.5 with mixed cultures, which is quite unusual and explains the ability of the present biocatalyst to remove alkylbenzenes with high efficiency in biofilters under acidic conditions.     

The mechanism of the converter domain rotation in the recovery stroke of myosin motor protein

Upon ATP binding, myosin motor protein is found in two alternative conformations, prerecovery state M* and postrecovery state M**. The transition from one state to the other, known as the recovery stroke, plays a key role in the myosin functional cycle. Despite much recent research, the microscopic details of this transition remain elusive. A critical step in the recovery stroke is the rotation of the converter domain from “up” position in prerecovery state to “down” position in postrecovery state that leads to the swing of the lever arm attached to it. In this work, we demonstrate that the two rotational states of the converter domain are determined by the interactions within a small structural motif in the force‐generating region of the protein that can be accurately modeled on computers using atomic representation and explicit solvent. Our simulations show that the transition between the two states is controlled by a small helix (SH1) located next to the relay helix and relay loop. A small translation in the position of SH1 away from the relay helix is seen to trigger the transition from “up” state to “down” state. The transition is driven by a cluster of hydrophobic residues I687, F487, and F506 that make significant contributions to the stability of both states. The proposed mechanism agrees well with the available structural and mutational studies. Proteins 2012; © 2012 Wiley Periodicals, Inc.     

Electrophoretic profiles of cyanobacterial membrane polypeptides showing heme-dependent peroxidase activity

The photosynthetic membranes of Anacystis nidulans R2 were examined electrophoretically following solubilization with lithium dodecyl sulfate. Electrophoresis yielded six prominent chlorophyll-containing bands. In addition, five polypeptides were observed which possessed heme-dependent peroxidase activity, monitored by incubating gels with 3,3′,5,5′-tetramethylbenzidine plus hydrogen peroxide. One such polypeptide, at 105 kdaltons, was removed by repeated washing of the membranes. Four remaining peroxidase-active polypeptides were observed at 7.2, 13.5, 18.5 and 33 kdaltons. Further examination of these four polypeptides yielded the following results. (1) The mobility of the 33 kdalton polypeptide was altered from 29 to 33 kdaltons upon heating (70°C) during membrane solubilization. (2) All four polypeptides showed stable heme-protein associations in the presence of 8 M urea; however, in the presence of urea, alterations in protein mobility were observed for each poly-peptide and only two (at 13.5 and 33 kdaltons) showed peroxidase activity following heating (70°C) during membrane solubilization. (3) The presence of thiols during membrane solubilization at 0°C was required to observe peroxidase activity at 7.2 kdaltons. These results, when compared to known properties of isolated cytochromes, suggest that the four polypeptides characterized here correspond to the subunits of photosynthetic cytochromes. Electrophoretic assessment of maize mutants lacking cytochrome f and b₆ activity supports this suggestion.     

Redox chain and energy transduction in chromatophores from Rhodopseudomonas capsulata cells grown anaerobically in the dark on glucose and dimethyl sulfoxide

Membranes from cells of Rhodopseudomonas capsulata grown anaerobically in the dark on glucose plus dimethyl sulfoxide differ from those obtained from photoheterotrophically grown cells in several ways: (a) there are qualitative and quantitative variations in the cytochrome composition; (b) electron-transport rates are unusually low in the cytochrome b to cytochrome c region; (c) light-induced ATP synthesis is dependent on the ability of the alternate respiratory pathway to maintain the Q₁₀-cytochrome b complex in a partially oxidized state; (d) a non-energy-conserving NADH-dehydrogenase activity dominates the respiratory activity. In addition, data obtained with both wild-type and mutant cells that contain altered electron-transport systems tend to exclude a role of the redox chain as ATP-producing machinery during anaerobic/dark growth.     

Pressure effects on the apparent viscosity of artificial dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine membranes using intramolecular excimer probe

Emission spectroscopy of intramolecular excimer probes allows the determination of ‘equivalent viscosity’ of membranes. While increasing the pressure on artificial membrane suspensions, variations in viscosity — essentially related to an increase in the order parameter in the membranes — are observed. In the case of mixed phospholipids, the effect of pressure is amplified, probably due to the existence of holes on the molecular scale between the two lipidic layers.     

Exchange of phosphatidylcholine between small unilamellar liposomes and human plasma high-density lipoprotein involves exclusively the phospholipid in the outer monolayer of the liposomal membrane

By making use of the capacity of phospholipase A₂ to degrade selectively the phospholipid in the outer half of the lipid bilayer of small unilamellar phospholipid/cholesterol vesicles without affecting the retention of a vesicle-encapsulated solute, we demonstrated that the exchange of phosphatidylcholine between such vesicles and human high density lipoprotein involves exclusively the phosphatidylcholine present in the outer monolayer of the vesicle membrane.     

Construction of a physical map of the 45 kb photosynthetic gene cluster of Rhodobacter sphaeroides

1) A number of overlapping clones have been isolated from a Rhodobacter sphaeroides gene bank. Following conjugative gene transfer from Escherichia coli these clones restore a wild type phenotype to several mutants unable to synthesise bacteriochlorophyll. 2) The insert DNA was analysed by restriction mapping and together the clones form the basis of the first restriction map of the 45 kb photosynthetic gene cluster of Rb. sphaeroides. 3) This cluster is bounded on one side by puh A encoding the reaction centre H polypeptide and on the other by the puf operon encoding reaction centre L and M apoproteins and light harvesting LH1 and polypeptides. 4) DNA fragments from the 45 kb cluster were used to probe genomic DNA from other photosynthetic bacteria. Using heterologous hybridisation conditions, a significant degree of homology is shown between Rb. sphaeroides and these other bacteria, suggesting close evolutionary links with Rb. capsulatus in particular.