Calcium binding to a disordered domain of a type III-secreted protein from a coral pathogen promotes secondary structure formation and catalytic activity

Strains of the Gram-negative bacterium Vibrio coralliilyticus cause the bleaching of corals due to decomposition of symbiotic microalgae. The V. coralliilyticus strain ATCC BAA-450 (Vc450) encodes a type III secretion system (T3SS). The gene cluster also encodes a protein (locus tag VIC_001052) with sequence homology to the T3SS-secreted nodulation proteins NopE1 and NopE2 of Bradyrhizobium japonicum (USDA110). VIC_001052 has been shown to undergo auto-cleavage in the presence of Ca2+ similar to the NopE proteins. We have studied the hitherto unknown secondary structure, Ca2+-binding affinity and stoichiometry of the "metal ion-inducible autocleavage" (MIIA) domain of VIC_001052 which does not possess a classical Ca2+-binding motif. CD and fluorescence spectroscopy revealed that the MIIA domain is largely intrinsically disordered. Binding of Ca2+ and other di- and trivalent cations induced secondary structure and hydrophobic packing after partial neutralization of the highly negatively charged MIIA domain. Mass spectrometry and isothermal titration calorimetry showed two Ca2+-binding sites which promote structure formation with a total binding enthalpy of -110 kJ mol(-1) at a low micromolar K-d. Putative binding motifs were identified by sequence similarity to EF-hand domains and their structure analyzed by molecular dynamics simulations. The stoichiometric Ca2+-dependent induction of structure correlated with catalytic activity and may provide a "host-sensing" mechanism that is shared among pathogens that use a T3SS for efficient secretion of disordered proteins

The Effect of Changing the Contraction Mode During Resistance Training on mTORC1 Signaling and Muscle Protein Synthesis

Acute resistance exercise (RE) increases muscle protein synthesis (MPS) via activation of mechanistic target of rapamycin complex (mTORC), and chronic resistance exercise training (RT) results in skeletal muscle hypertrophy. Although MPS in response to RE is blunted over time during RT, no effective restorative strategy has been identified. Since eccentric muscle contraction (EC) has the potential to strongly stimulate mTORC1 activation and MPS, changing the muscle contraction mode to EC might maintain the MPS response to RE during chronic RT. Male rats were randomly divided into RE (1 bout of RE) and RT (13 bouts of RE) groups. Additionally, each group was subdivided into isometric contraction (IC) and EC subgroups. The RE groups performed acute, unilateral RE using IC or EC. The RT groups performed 12 bouts of unilateral RE using IC. For bout 13, the RT-IC subgroup performed a further IC bout, while the RT-EC subgroup changed to EC. All muscle contractions were induced by percutaneous electrical stimulation. Muscle samples were obtained at 6 h post exercise in all groups. After the 1st RE bout, the EC group showed significantly higher p70S6K Thr389 phosphorylation than the IC group. However, the phosphorylation of other mTORC1-associated proteins (4E-BP1 and ribosomal protein S6) and the MPS response did not differ between the contraction modes. After the 13th bout of RE, mTORC1 activation and the MPS response were significantly blunted as compared with the 1st bout of RE. Changing from IC to EC did not improve these responses. In conclusion, changing the contraction mode to EC does not reinvigorate the blunted mTORC1 activation and MPS in response to RE during chronic RT

Task Attention Facilitates Learning of Task-Irrelevant Stimuli

Attention plays a fundamental role in visual learning and memory. One highly established principle of visual attention is that the harder a central task is, the more attentional resources are used to perform the task and the smaller amount of attention is allocated to peripheral processing because of limited attention capacity. Here we show that this principle holds true in a dual-task setting but not in a paradigm of task-irrelevant perceptual learning. In Experiment 1, eight participants were asked to identify either bright or dim number targets at the screen center and to remember concurrently presented scene backgrounds. Their recognition performances for scenes paired with dim/hard targets were worse than those for scenes paired with bright/easy targets. In Experiment 2, eight participants were asked to identify either bright or dim letter targets at the screen center while a task-irrelevant coherent motion was concurrently presented in the background. After five days of training on letter identification, participants improved their motion sensitivity to the direction paired with hard/dim targets improved but not to the direction paired with easy/bright targets. Taken together, these results suggest that task-irrelevant stimuli are not subject to the attentional control mechanisms that task-relevant stimuli abide

2-Phosphonobutane-1,2,4,-Tricarboxylic Acid (PBTC): pH-Dependent Behavior Studied by Means of Multinuclear NMR Spectroscopy

Although 2-phosphonobutane-1,2,4,-tricarboxylic acid, PBTC, has manifold industrial applications, relevant and reliable data on the protonation of PBTC are poor. However, these data are critical parameters for ascertaining PBTC speciation, especially with regard to a sound structural and thermodynamic characterization of its metal ion complexes. A rigorous evaluation of pH-dependent 1H, 13C, and 31P chemical shifts along with accessible scalar spin–spin coupling constants (J) was performed in order to determine the pKa values of PBTC in 0.5 molal NaCl aqueous solution by means of nuclear magnetic resonance (NMR) spectroscopy. The phosphonate group revealed pKa values of 0.90 ± 0.02 and 9.79 ± 0.02, and the pKa values associated with the carboxylic groups are 3.92 ± 0.02, 4.76 ± 0.03, and 6.13 ± 0.03. Supported by DFT-calculated structures revealing strong intramolecular hydrogen bonding, the sequence of deprotonation could be unambiguously determined

DNA Based Formation of Nanodisc-Stacks

We describe here the formation of multimers of membrane-scaffolding protein MSP1D1-bounded nanodiscs using the thiol reactivity of engineered cysteines. The mutated positions N42 and K163 in MSP1D1 were chosen to support chemical modification as evidenced by fluorescent labeling with pyrene. The direct disulphide bond formation of nanodiscs formed by the MSP1D1_N42C variant led to dimers and trimers with low yield. In contrast, transmission electron microscopy revealed that the attachment of oligonucleotides to the engineered cysteines of MSP1D1 allowed the growth of submicron-sized tracts of stacked nanodiscs through the hybridization of nanodisc populations carrying complementary strands and a flexible spacer