Membrane proteins depend on complex translocation machineries for insertion into target membranes. Although it has long been known that an abundance of nonpolar residues in transmembrane helices is the principal criterion for membrane insertion, the specific sequence-coding for transmembrane helices has not been identified. By challenging the endoplasmic reticulum Sec61 translocon with an extensive set of designed polypeptide segments, we have determined the basic features of this code, including a 'biological' hydrophobicity scale. We find that membrane insertion depends strongly on the position of polar residues within transmembrane segments, adding a new dimension to the problem of predicting transmembrane helices from amino acid sequences. Our results indicate that direct protein-lipid interactions are critical during translocon-mediated membrane insertion.
Transmembrane alpha-helices in integral membrane proteins are recognized co-translationally and inserted into the membrane of the endoplasmic reticulum by the Sec61 translocon. A full quantitative description of this phenomenon, linking amino acid sequence to membrane insertion efficiency, is still lacking. Here, using in vitro translation of a model protein in the presence of dog pancreas rough microsomes to analyse a large number of systematically designed hydrophobic segments, we present a quantitative analysis of the position-dependent contribution of all 20 amino acids to membrane insertion efficiency, as well as of the effects of transmembrane segment length and flanking amino acids. The emerging picture of translocon-mediated transmembrane helix assembly is simple, with the critical sequence characteristics mirroring the physical properties of the lipid bilayer.
Abstract. In this paper, we propose a new block cipher HIGHT with 64-bit block length and 128-bit key length. It provides low-resource hardware implementation, which is proper to ubiquitous computing device such as a sensor in USN or a RFID tag. HIGHT does not only consist of simple operations to be ultra-light but also has enough security as a good encryption algorithm. Our hardware implementation of HIGHT requires 3048 gates on 0.25 µm technology.
It is often difficult to produce eukaryotic membrane proteins in large quantities, which is a major obstacle for analyzing their biochemical and structural features. To date, yeast has been the most successful heterologous overexpression system in producing eukaryotic membrane proteins for highresolution structural studies. For this reason, we have developed a protocol for rapidly screening and purifying eukaryotic membrane proteins in the yeast Saccharomyces cerevisiae. Using this protocol, in 1 week many genes can be rapidly cloned by homologous recombination into a 2 μGFP-fusion vector and their overexpression potential determined using whole-cell and in-gel fluorescence. The quality of the overproduced eukaryotic membrane protein-GFP fusions can then be evaluated over several days using confocal microscopy and fluorescence size-exclusion chromatography (FSEC). This protocol also details the purification of targets that pass our quality criteria, and can be scaled up for a large number of eukaryotic membrane proteins in either an academic, structural genomics or commercial environment.
Synaptic cell adhesion molecules (CAMs) regulate synapse formation through their trans-synaptic and heterophilic adhesion. Here we show that postsynaptic netrin-G ligand (NGL) CAMs associate with netrin-G CAMs in an isoform-specific manner and, through their cytosolic tail, with the abundant postsynaptic scaffold postsynaptic density-95 (PSD-95). Overexpression of NGL-2 in cultured rat neurons increased the number of PSD-95-positive dendritic protrusions. NGL-2 located on heterologous cells or beads induced functional presynaptic differentiation in contacting neurites. Direct aggregation of NGL-2 on the surface membrane of dendrites induced the clustering of excitatory postsynaptic proteins. Competitive inhibition by soluble NGL-2 reduced the number of excitatory synapses. NGL-2 knockdown reduced excitatory, but not inhibitory, synapse numbers and currents. These results suggest that NGL regulates the formation of excitatory synapses.
Auditory fear memory is thought to be maintained by fear conditioning-induced potentiation of synaptic efficacy, which involves enhanced expression of surface AMPA receptor (AMPAR) at excitatory synapses in the lateral amygdala (LA). Depotentiation, reversal of conditioning-induced potentiation, has been proposed as a cellular mechanism for fear extinction; however, a direct link between depotentiation and extinction has not yet been tested. To address this issue, we applied both ex vivo and in vivo approaches to rats in which fear memory had been consolidated. A unique form of depotentiation reversed conditioning-induced potentiation at thalamic input synapses onto the LA (T-LA synapses) ex vivo. Extinction returned the enhanced T-LA synaptic efficacy observed in conditioned rats to baseline and occluded the depotentiation. Consistently, extinction reversed conditioning-induced enhancement of surface expression of AMPAR subunits in LA synaptosomal preparations. A GluR2-derived peptide that blocks regulated AMPAR endocytosis inhibited depotentiation, and microinjection of a cell-permeable form of the peptide into the LA attenuated extinction. Our results are consistent with the use of depotentiation to weaken potentiated synaptic inputs onto the LA during extinction and provide strong evidence that AMPAR removal at excitatory synapses in the LA underlies extinction.lateral amygdala ͉ fear conditioning ͉ AMPA receptor ͉ endocytosis T he cortical and thalamic input synapses onto the lateral amygdala (LA) (C-LA and T-LA synapses, respectively) carry auditory information from the auditory cortex and auditory thalamus onto the LA, respectively (1). Long-term potentiation (LTP; an in vitro model of memory) (2)-like changes in these pathways are thought to underlie both the encoding and consolidation of auditory fear memory (3-8). The results of a recent study suggest that long-term retention of conditioning-induced potentiation at excitatory synapses in the LA is a critical requirement for consolidated fear memory within the LA (7, 9). Also, LTP requiring the synaptic delivery of AMPA receptors (AMPARs) at excitatory synapses in the LA appears to be necessary for establishing consolidated fear memory (6,8,10). Conditioning-induced potentiation and auditory fear memory encoded in the LA have been shown to be consolidated within 24 h after fear conditioning (5, 7, 11). Moreover, auditory fear memory appears to be maintained in the LA across the adult lifetime of rats (12). Thus, consolidation of auditory fear memory encoded in the LA is rapid and localized, unlike hippocampus-dependent memory, which involves slow and distributed consolidation processes (13).In the present study, we tested the hypothesis that depotentiation of conditioning-induced potentiation at excitatory synapses in the LA underlies extinction of consolidated fear memory. Synaptic weights were monitored ex vivo by using whole-cell (or field potential) recordings in amygdala slices prepared from behaviortrained rats. Results Extinction of Consolidated ...
Meeting NASA's N+3 goals requires a fundamental shift in approach to aircraft and engine design. Material and design improvements allow higher pressure and higher temperature core engines which improve the thermal efficiency. Propulsive efficiency, the other half of the overall efficiency equation, however, is largely determined by the fan pressure ratio (FPR). Lower FPR increases propulsive efficiency, but also dramatically reduces fan shaft speed through the combination of larger diameter fans and reduced fan tip speed limits. The result is that below an FPR of 1.5 the maximum fan shaft speed makes direct drive turbines problematic. However, it is the low pressure ratio fans that allow the improvement in propulsive efficiency which, along with improvements in thermal efficiency in the core, contributes strongly to meeting the N+3 goals for fuel burn reduction. The lower fan exhaust velocities resulting from lower FPRs are also key to meeting the aircraft noise goals. Adding a gear box to the standard turbofan engine allows acceptable turbine speeds to be maintained. However, development of a 50,000+ hp gearbox required by fans in a large twin engine transport aircraft presents an extreme technical challenge, therefore another approach is needed. This paper presents a propulsion system which transmits power from the turbine to the fan electrically rather than mechanically. Recent and anticipated advances in high temperature superconducting generators, motors, and power lines offer the possibility that such devices can be used to transmit turbine power in aircraft without an excessive weight penalty. Moving to such a power transmission system does more than provide better matching between fan and turbine shaft speeds. The relative ease with which electrical power can be distributed throughout the aircraft opens up numerous other possibilities for new aircraft and propulsion configurations and modes of operation. This paper discusses a number of these new possibilities. The Boeing N2 hybrid-wing-body (HWB) is used as a baseline aircraft for this study. The two pylon mounted conventional turbofans are replaced by two wing-tip mounted turboshaft engines, each driving a superconducting generator. Both generators feed a common electrical bus which distributes power to an array of superconducting motor-driven fans in a continuous nacelle centered along the trailing edge of the upper surface of the wing-body. A key finding was that traditional inlet performance methodology has to be modified when most of the air entering the inlet is boundary layer air. A very thorough and detailed propulsion/airframe integration (PAI) analysis is required at the very beginning of the design process since embedded engine inlet performance must be based on conditions at the inlet lip rather than freestream conditions. Examination of a range of fan pressure ratios yielded a minimum Thrust-specific-fuel-consumption (TSFC) at the aerodynamic design point of the vehicle (31,000 ft /Mach 0.8) between 1.3 and 1.35 FPR. We deduced that this wa...
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