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1
Silberberg, Shai D., Tsg-Hui Chang, and Kenton J. Swartz. "Secondary Structure and Gating Rearrangements of Transmembrane Segments in Rat P2X4 Receptor Channels." Journal of General Physiology 125, no. 4 (2005): 347–59. http://dx.doi.org/10.1085/jgp.200409221.
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P2X receptors are cation selective channels that are activated by extracellular nucleotides. These channels are likely formed by three identical or related subunits, each having two transmembrane segments (TM1 and TM2). To identify regions that undergo rearrangement during gating and to probe their secondary structure, we performed tryptophan scanning mutagenesis on the two putative TMs of the rat P2X4 receptor channel. Mutant channels were expressed in Xenopus oocytes, concentration–response relationships constructed for ATP, and the EC50 estimated by fitting the Hill equation to the data. Of the 22 mutations in TM1 and 24 in TM2, all but one in TM1 and seven in TM2 result in functional channels. Interestingly, the majority of the functional mutants display an increased sensitivity to ATP, and in general these perturbations are more pronounced for TM2 when compared with TM1. For TM1 and for the outer half of TM2, the perturbations are consistent with these regions adopting α-helical secondary structures. In addition, the greatest perturbations in the gating equilibrium occur for mutations near the outer ends of both TM1 and TM2. Surface biotinylation experiments reveal that all the nonfunctional mutants traffic to the surface membrane at levels comparable to the WT channel, suggesting that these mutations likely disrupt ion conduction or gating. Taken together, these results suggest that the outer parts of TM1 and TM2 are helical and that they move during activation. The observation that the majority of nonconducting mutations are clustered toward the inner end of TM2 suggests a critical functional role for this region.
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Stopp, Marius, Philipp A. Steinmetz, and Gottfried Unden. "Properties of transmembrane helix TM1 of the DcuS sensor kinase of Escherichia coli, the stator for TM2 piston signaling." Biological Chemistry 402, no. 10 (2021): 1239–46. http://dx.doi.org/10.1515/hsz-2021-0254.
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Abstract The sensor kinase DcuS of Escherichia coli perceives extracellular fumarate by a periplasmic PASP sensor domain. Transmembrane (TM) helix TM2, present as TM2-TM2′ homo-dimer, transmits fumarate activation in a piston-slide across the membrane. The second TM helix of DcuS, TM1, is known to lack piston movement. Structural and functional properties of TM1 were analyzed. Oxidative Cys-crosslinking (CL) revealed homo-dimerization of TM1 over the complete membrane, but only the central part showed α-helical +3/+4 spacing of the CL maxima. The GALLEX bacterial two-hybrid system indicates TM1/TM1′ interaction, and the presence of a TM1-TM1′ homo-dimer is suggested. The peripheral TM1 regions presented CL in a spacing atypical for α-helical arrangement. On the periplasmic side the deviation extended over 11 AA residues (V32-S42) between the α-helical part of TM1 and the onset of PASP. In the V32-S42 region, CL efficiency decreased in the presence of fumarate. Therefore, TM1 exists as a homo-dimer with α-helical arrangement in the central membrane region, and non-α-helical arrangement in the connector to PASP. The fumarate induced structural response in the V32-S42 region is suggested to represent a structural adaptation to the shift of TM2 in the TM1-TM1′/TM2-TM2′ four-helical bundle.
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Monzel, Christian, and Gottfried Unden. "Transmembrane signaling in the sensor kinase DcuS ofEscherichia coli: A long-range piston-type displacement of transmembrane helix 2." Proceedings of the National Academy of Sciences 112, no. 35 (2015): 11042–47. http://dx.doi.org/10.1073/pnas.1507217112.
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The C4-dicarboxylate sensor kinase DcuS is membrane integral because of the transmembrane (TM) helices TM1 and TM2. Fumarate-induced movement of the helices was probed in vivo by Cys accessibility scanning at the membrane–water interfaces after activation of DcuS by fumarate at the periplasmic binding site. TM1 was inserted with amino acid residues 21–41 in the membrane in both the fumarate-activated (ON) and inactive (OFF) states. In contrast, TM2 was inserted with residues 181–201 in the OFF state and residues 185–205 in the ON state. Replacement of Trp 185 by an Arg residue caused displacement of TM2 toward the outside of the membrane and a concomitant induction of the ON state. Results from Cys cross-linking of TM2/TM2′ in the DcuS homodimer excluded rotation; thus, data from accessibility changes of TM2 upon activation, either by ligand binding or by mutation of TM2, and cross-linking of TM2 and the connected region in the periplasm suggest a piston-type shift of TM2 by four residues to the periplasm upon activation (or fumarate binding). This mode of function is supported by the suggestion from energetic calculations of two preferred positions for TM2 insertion in the membrane. The shift of TM2 by four residues (or 4–6 Å) toward the periplasm upon activation is complementary to the periplasmic displacement of 3–4 Å of the C-terminal part of the periplasmic ligand-binding domain upon ligand occupancy in the citrate-binding domain in the homologous CitA sensor kinase.
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TAKAI, Akira, Katsunori TSUBOI, Masayoshi KOYASU, and Minoru ISOBE. "Effects of modification of the hydrophobic C-1–C-16 segment of tautomycin on its affinity to type-1 and type-2A protein phosphatases." Biochemical Journal 350, no. 1 (2000): 81–88. http://dx.doi.org/10.1042/bj3500081.
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Among the naturally occurring toxins that are known to have specific inhibitory effects on type-1 and type-2A protein phosphatases (PP1 and PP2A), tautomycin (TM) is unique in that it exhibits significantly higher affinity to PP1 than to PP2A. The ratio of the dissociation constant for the PP1–TM interaction to that for the PP2A–TM interaction (the PP1/PP2A ratio) is 0.01–0.03. The aim of the present study was to evaluate the possible contributions of the C-1–C-16 segment of TM to the affinity characteristics of the toxin. The relatively hydrophobic segment contains a spiroketal motif whose enantiomeric form is present in okadaic acid (OA), which exhibits exceedingly higher affinity to PP2A than to PP1. We therefore synthesized two TM analogues: TM1 in which the side chains of the spiroketal motif of TM were removed but its absolute configuration was retained, and TM2 in which the spiroketal motif of TM1 was replaced with its enantiomeric form. The effects of TM, TM1 and TM2 on the activities of the native catalytic subunits of PP1 (PP1C) and PP2A and a recombinant γ isoform of PP1 (PP1γ) were examined. The PP1/PP2A ratio determined thereby was 0.2–0.5 for TM1 and 5–10 for TM2. Both the presence of the side chains and the stereochemistry of the spiroketal moieties may be major determining factors for the affinity characteristics of TM. We also show that a monoclonal antibody raised against OA binds to TM2 albeit with much lower affinity than to OA, whereas it exhibits no measurable affinities to TM and TM1.
5
Matenchuk, Brittany A., Marina James, Rachel J. Skow, et al. "Longitudinal study of cerebral blood flow regulation during exercise in pregnancy." Journal of Cerebral Blood Flow & Metabolism 40, no. 11 (2019): 2278–88. http://dx.doi.org/10.1177/0271678x19889089.
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Cerebrovascular adaptation to pregnancy is poorly understood. We sought to assess cerebrovascular regulation in response to visual stimulation, hypercapnia and exercise across the three trimesters of pregnancy. Using transcranial Doppler (TCD) ultrasound, middle and posterior cerebral artery mean blood velocities (MCAvmean and PCAvmean) were measured continuously at rest and in response to (1) visual stimulation to assess neurovascular coupling (NVC); (2) a modified Duffin hyperoxic CO2 rebreathe test, and (3) an incremental cycling exercise test to volitional fatigue in non-pregnant ( n = 26; NP) and pregnant women (first trimester [ n = 13; TM1], second trimester [ n = 21; TM2], and third trimester [ n = 20; TM3]) in total 47 women. At rest, MCAvmean and PETCO2 were lower in TM2 compared to NP. PCAvmean was lower in TM2 but not TM1 or TM3 compared to NP. Cerebrovascular reactivity in MCAvmean and PCAvmean during the hypercapnic rebreathing test was not different between pregnant and non-pregnant women. MCAvmean continued to increase over the second half of the exercise test in TM2 and TM3, while it decreased in NP due to differences in ΔPETCO2 between groups. Pregnant women experienced a delayed decrease in MCAvmean in response to maximal exercise compared to non-pregnant controls which was explained by CO2 reactivity and PETCO2 level.
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Liu, Guoxia, Xiaowei Niu, Roland S. Wu та ін. "Location of modulatory β subunits in BK potassium channels". Journal of General Physiology 135, № 5 (2010): 449–59. http://dx.doi.org/10.1085/jgp.201010417.
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Large-conductance voltage- and calcium-activated potassium (BK) channels contain four pore-forming α subunits and four modulatory β subunits. From the extents of disulfide cross-linking in channels on the cell surface between cysteine (Cys) substituted for residues in the first turns in the membrane of the S0 transmembrane (TM) helix, unique to BK α, and of the voltage-sensing domain TM helices S1–S4, we infer that S0 is next to S3 and S4, but not to S1 and S2. Furthermore, of the two β1 TM helices, TM2 is next to S0, and TM1 is next to TM2. Coexpression of α with two substituted Cys’s, one in S0 and one in S2, and β1 also with two substituted Cys’s, one in TM1 and one in TM2, resulted in two αs cross-linked by one β. Thus, each β lies between and can interact with the voltage-sensing domains of two adjacent α subunits.
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Popova, Maya, Larry Rodriguez, James R. Trudell, et al. "Residues in Transmembrane Segments of the P2X4 Receptor Contribute to Channel Function and Ethanol Sensitivity." International Journal of Molecular Sciences 21, no. 7 (2020): 2471. http://dx.doi.org/10.3390/ijms21072471.
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Mouse models of alcohol use disorder (AUD) revealed purinergic P2X4 receptors (P2X4Rs) as a promising target for AUD drug development. We have previously demonstrated that residues at the transmembrane (TM)–ectodomain interface and within the TM1 segment contribute to the formation of an ethanol action pocket in P2X4Rs. In the present study, we tested the hypothesis that there are more residues in TM1 and TM2 segments that are important for the ethanol sensitivity of P2X4Rs. Using site-directed mutagenesis and two electrode voltage-clamp electrophysiology in Xenopus oocytes, we found that arginine at position 33 (R33) in the TM1 segment plays a role in the ethanol sensitivity of P2X4Rs. Molecular models in both closed and open states provided evidence for interactions between R33 and aspartic acid at position 354 (D354) of the neighboring TM2 segment. The loss of ethanol sensitivity in mixtures of wild-type (WT) and reciprocal single mutants, R33D:WT and D354R:WT, versus the WT-like response in R33D-D354R:WT double mutant provided further support for this interaction. Additional findings indicated that valine at TM1 position 49 plays a role in P2X4R function by providing flexibility/stability during channel opening. Collectively, these findings identified new activity sites and suggest the importance of TM1-TM2 interaction for the function and ethanol sensitivity of P2X4Rs.
8
Li, Jie, Jianli Guo, Xiaomin Ou, Mingfeng Zhang, Yuezhou Li, and Zhenfeng Liu. "Mechanical coupling of the multiple structural elements of the large-conductance mechanosensitive channel during expansion." Proceedings of the National Academy of Sciences 112, no. 34 (2015): 10726–31. http://dx.doi.org/10.1073/pnas.1503202112.
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The prokaryotic mechanosensitive channel of large conductance (MscL) is a pressure-relief valve protecting the cell from lysing during acute osmotic downshock. When the membrane is stretched, MscL responds to the increase of membrane tension and opens a nonselective pore to about 30 Å wide, exhibiting a large unitary conductance of ∼3 nS. A fundamental step toward understanding the gating mechanism of MscL is to decipher the molecular details of the conformational changes accompanying channel opening. By applying fusion-protein strategy and controlling detergent composition, we have solved the structures of an archaeal MscL homolog from Methanosarcina acetivorans trapped in the closed and expanded intermediate states. The comparative analysis of these two new structures reveals significant conformational rearrangements in the different domains of MscL. The large changes observed in the tilt angles of the two transmembrane helices (TM1 and TM2) fit well with the helix-pivoting model derived from the earlier geometric analyses based on the previous structures. Meanwhile, the periplasmic loop region transforms from a folded structure, containing an ω-shaped loop and a short β-hairpin, to an extended and partly disordered conformation during channel expansion. Moreover, a significant rotating and sliding of the N-terminal helix (N-helix) is coupled to the tilting movements of TM1 and TM2. The dynamic relationships between the N-helix and TM1/TM2 suggest that the N-helix serves as a membrane-anchored stopper that limits the tilts of TM1 and TM2 in the gating process. These results provide direct mechanistic insights into the highly coordinated movement of the different domains of the MscL channel when it expands.
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Wotring, Virginia E., and David S. Weiss. "Charge Scan Reveals an Extended Region at the Intracellular End of the GABA Receptor Pore that Can Influence Ion Selectivity." Journal of General Physiology 131, no. 1 (2007): 87–97. http://dx.doi.org/10.1085/jgp.200609701.
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Selective permeability is a fundamental property of ion channels. The Cys-loop receptor superfamily is composed of both excitatory (ACh, 5-HT) and inhibitory (GABA, glycine) neurotransmitter-operated ion channels. In the GABA receptor, it has been previously shown that the charge selectivity of the integral pore can be altered by a single mutation near the intracellular end of the second transmembrane-spanning domain (TM2). We have extended these findings and now show that charge selectivity of the anionic ρ1 GABA receptor can be influenced by the introduction of glutamates, one at a time, over an 8–amino acid stretch (−2′ to 5′) in the proposed intracellular end of TM2 and the TM1–TM2 intracellular linker. Depending on the position, glutamate substitutions in this region produced sodium to chloride permeability ratios (PNa+/Cl−) varying from 0.64 to 3.4 (wild type PNa+/Cl− = 0). In addition to providing insight into the mechanism of ion selectivity, this functional evidence supports a model proposed for the homologous nicotinic acetylcholine receptor in which regions of the protein, in addition to TM2, form the ion pathway.
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Pippel, Anja, Michaela Stolz, Ronja Woltersdorf, Achim Kless, Günther Schmalzing, and Fritz Markwardt. "Localization of the gate and selectivity filter of the full-length P2X7 receptor." Proceedings of the National Academy of Sciences 114, no. 11 (2017): E2156—E2165. http://dx.doi.org/10.1073/pnas.1610414114.
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The P2X7 receptor (P2X7R) belongs to the P2X family of ATP-gated cation channels. P2X7Rs are expressed in epithelial cells, leukocytes, and microglia, and they play important roles in immunological and inflammatory processes. P2X7Rs are obligate homotrimers, with each subunit having two transmembrane helices, TM1 and TM2. Structural and functional data regarding the P2X2 and P2X4 receptors indicate that the central trihelical TM2 bundle forms the intrinsic transmembrane channel of P2X receptors. Here, we studied the accessibility of single cysteines substituted along the pre-TM2 and TM2 helix (residues 327–357) of the P2X7R using as readouts (i) the covalent maleimide fluorescence accessibility of the surface-bound P2X7R and (ii) covalent modulation of macroscopic and single-channel currents using extracellularly and intracellularly applied methanethiosulfonate (MTS) reagents. We found that the channel opening extends from the pre-TM2 region through the outer half of the trihelical TM2 channel. Covalently adducted MTS ethylammonium+ (MTSEA+) strongly increased the probability that the channel was open by delaying channel closing of seven of eight responsive human P2X7R (hP2X7R) mutants. Structural modeling, as supported by experimental probing, suggested that resulting intraluminal hydrogen bonding interactions stabilize the open-channel state. The additional decrease in single-channel conductance by MTSEA+ in five of seven positions identified Y336, S339, L341C, Y343, and G345 as the narrowest part of the channel lumen. The gate and ion-selectivity filter of the P2X7R could be colocalized at and around residue S342. None of our results provided any evidence for dilation of the hP2X7R channel on sustained stimulation with ATP4−.
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Nakazawa, Youya, Shigeo Sato, Mikihiko Naito, et al. "Tetraspanin family member CD9 inhibits Aggrus/podoplanin-induced platelet aggregation and suppresses pulmonary metastasis." Blood 112, no. 5 (2008): 1730–39. http://dx.doi.org/10.1182/blood-2007-11-124693.
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Abstract CD9 has been reported to play a role in tumor metastasis suppression. However, it is not fully understood how CD9 affects the hematogenous spread of tumor cells. To clarify a new mechanism (or mechanisms), we generated HT1080 cells that had been transfected with a CD9-expressing plasmid. Ectopic expression of CD9 in HT1080 cells actually reduced their metastatic ability. CD9 expression reduced lung retention and platelet ag-gregation activity of the transfectants. Because HT1080 cells express the metastasis-promoting, platelet aggregation-inducing factor Aggrus/podoplanin on their surface, we examined the relationship between CD9 and Aggrus. We discovered that CD9 formed a complex with Aggrus via transmembrane domains 1 and 2 (TM1 and TM2) of CD9. Investigation of the interaction revealed that each CD9 and Aggrus interacted homophilically, and that they colocalized in low-density membrane fractions. Deleting TM1 and TM2 attenuated the ability of CD9 to interact homophilically or to localize in low-density membrane fractions. The expression of CD9–wild-type (WT), but not CD9 lacking TM1 and TM2, attenuated the platelet aggregation and metastasis induced by forced expression of Aggrus in CHO cells. Therefore, CD9 may act as a metastasis suppressor, at least in part, by neutralizing Aggrus-mediated platelet aggregation.
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Salman, Ammar H., and Sarmad Alhadethy. "Antibacterial Residues in Some Imported Chickens to Iraq." NeuroQuantology 19, no. 8 (2021): 145–48. http://dx.doi.org/10.14704/nq.2021.19.8.nq21126.
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In this study three of trademarks of frozen chickens imported to Iraq were chosen to study the antibacterials residues in the chicken meat. The three trade marks were TM1, TM2 and TM3. Three chickens of each trade mark were bought from local market of Baghdad, from each chick a piece of muscle was cut from the thigh and the chest and from each piece four samples were tested for antimicrobial residues by using four plate test (FTP). The results showed presence of antibacterials residues in 49% of tested samples. For TM2 and TM3 the positive samples were 62.5% and 83% respectively while no antibacterials residues detected for TM1. Concluded from this study the presence of antibacterials residues in imported chickens to Iraq.
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Leavitt, J., G. Latter, L. Lutomski, D. Goldstein, and S. Burbeck. "Tropomyosin isoform switching in tumorigenic human fibroblasts." Molecular and Cellular Biology 6, no. 7 (1986): 2721–26. http://dx.doi.org/10.1128/mcb.6.7.2721.
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We identified six tropomyosin (Tm) isoforms in diploid human fibroblasts. We used computerized microdensitometry of 2-dimensional protein profiles to measure the relative rates of synthesis and abundance of the individual Tm isoforms and actin, the two major structural constituents of microfilaments. In carcinogen-transformed human fibroblasts (HuT-14), the rates of synthesis of three Tm isoforms (Tm1, Tm2, and Tm6) were greatly decreased relative to normal diploid parental fibroblasts and to actin. In contrast, related nontumorigenic HuT fibroblasts which are "immortalized" and anchorage independent exhibited both slight down-regulation of Tm1 and Tm6 and 3.5-fold up-regulation of Tm3. Thus, Tm isoform switching from the predominance of the larger more avid Tm isoforms (Tm1, Tm2, Tm3, and Tm6) to the smaller, less avid Tm isoforms (Tm4 and Tm5) in microfilaments was a transformation-induced change correlated with tumorigenicity in human fibroblasts.
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Leavitt, J., G. Latter, L. Lutomski, D. Goldstein, and S. Burbeck. "Tropomyosin isoform switching in tumorigenic human fibroblasts." Molecular and Cellular Biology 6, no. 7 (1986): 2721–26. http://dx.doi.org/10.1128/mcb.6.7.2721-2726.1986.
Full textAbstract:
We identified six tropomyosin (Tm) isoforms in diploid human fibroblasts. We used computerized microdensitometry of 2-dimensional protein profiles to measure the relative rates of synthesis and abundance of the individual Tm isoforms and actin, the two major structural constituents of microfilaments. In carcinogen-transformed human fibroblasts (HuT-14), the rates of synthesis of three Tm isoforms (Tm1, Tm2, and Tm6) were greatly decreased relative to normal diploid parental fibroblasts and to actin. In contrast, related nontumorigenic HuT fibroblasts which are "immortalized" and anchorage independent exhibited both slight down-regulation of Tm1 and Tm6 and 3.5-fold up-regulation of Tm3. Thus, Tm isoform switching from the predominance of the larger more avid Tm isoforms (Tm1, Tm2, Tm3, and Tm6) to the smaller, less avid Tm isoforms (Tm4 and Tm5) in microfilaments was a transformation-induced change correlated with tumorigenicity in human fibroblasts.
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Takenaga, K., Y. Nakamura, S. Sakiyama, Y. Hasegawa, K. Sato, and H. Endo. "Binding of pEL98 protein, an S100-related calcium-binding protein, to nonmuscle tropomyosin." Journal of Cell Biology 124, no. 5 (1994): 757–68. http://dx.doi.org/10.1083/jcb.124.5.757.
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The cDNA coding for mouse fibroblast tropomyosin isoform 2 (TM2) was placed into a bacterial expression vector to produce a fusion protein containing glutathione-S-transferase (GST) and TM2 (GST/TM2). Glutathione-Sepharose beads bearing GST/TM2 were incubated with [35S]methionine-labeled NIH 3T3 cell extracts and the materials bound to the fusion proteins were analyzed to identify proteins that interact with TM2. A protein of 10 kD was found to bind to GST/TM2, but not to GST. The binding of the 10-kD protein to GST/TM2 was dependent on the presence of Ca2+ and inhibited by molar excess of free TM2 in a competition assay. The 10-kD protein-binding site was mapped to the region spanning residues 39-107 on TM2 by using several COOH-terminal and NH2-terminal truncation mutants of TM2. The 10-kD protein was isolated from an extract of NIH 3T3 cells transformed by v-Ha-ras by affinity chromatography on a GST/TM2 truncation mutant followed by SDS-PAGE and electroelution. Partial amino acid sequence analysis of the purified 10-kD protein, two-dimensional polyacrylamide gel analysis and a binding experiment revealed that the 10-kD protein was identical to a calcium-binding protein derived from mRNA named pEL98 or 18A2 that is homologous to S100 protein. Immunoblot analysis of the distribution of the 10-kD protein in Triton-soluble and -insoluble fractions of NIH 3T3 cells revealed that some of the 10-kD protein was associated with the Triton-insoluble cytoskeletal residue in a Ca(2+)-dependent manner. Furthermore, immunofluorescent staining of NIH 3T3 cells showed that some of the 10-kD protein colocalized with nonmuscle TMs in microfilament bundles. These results suggest that some of the pEL98 protein interacts with microfilament-associated nonmuscle TMs in NIH 3T3 cells.
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Cuéllar Lázaro, Carmen. "Los nombres propios y su tratamiento en traducción." Meta 59, no. 2 (2014): 360–79. http://dx.doi.org/10.7202/1027480ar.
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La présente étude vise à analyser les stratégies employées par différents traducteurs pour la traduction des noms propres dans les oeuvres littéraires. Sur la base de trois oeuvres de la littérature contemporaine en langue allemande, on analysera les traductions paires espagnoles de chaque traducteur, ce qui nous permettra, tout d’abord, de mettre en lumière les techniques utilisées dans la traduction des noms propres et leurs résultats, puis d’extrapoler à l’ensemble des trois doublets, pour voir s’il y a des aspects diachroniques qui distinguent les deuxièmes traductions (TM2) des premières traductions (TM1), c’est-à-dire s’il y a une relation entre la TM1 et TM2 analysées. L’analyse de la méthode employée par les six traducteurs est précédée d’une réflexion qui fournit des réponses à plusieurs questions liées aux propriétés idiosyncrasiques des noms propres et au traitement spécial en traduction.
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Borroto-Escuela, Wydra, Romero-Fernandez, et al. "A2AR Transmembrane 2 Peptide Administration Disrupts the A2AR-A2AR Homoreceptor but not the A2AR-D2R Heteroreceptor Complex: Lack of Actions on Rodent Cocaine Self-Administration." International Journal of Molecular Sciences 20, no. 23 (2019): 6100. http://dx.doi.org/10.3390/ijms20236100.
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It was previously demonstrated that rat adenosine A2AR transmembrane V peptide administration into the nucleus accumbens enhances cocaine self-administration through disruption of the A2AR-dopamine (D2R) heteroreceptor complex of this region. Unlike human A2AR transmembrane 4 (TM4) and 5 (TM5), A2AR TM2 did not interfere with the formation of the A2AR-D2R heteroreceptor complex in cellular models using BRET1 assay. A2AR TM2 was proposed to be part of the of the receptor interface of the A2AR homomer instead and was therefore tested in the current article for effects on rat cocaine self-administration using rat A2AR synthetic TM2 peptide bilaterally injected into the nucleus accumbens. The injected A2AR TM2 peptide failed to significantly counteract the inhibitory action of the A2AR agonist CGS 21680 (0.1 mg/Kg) on cocaine self-administration. In line with these results, the microinjected A2AR TM2 peptide did not reduce the number of proximity ligation assay blobs identifying A2AR-D2R heteroreceptor complexes in the nucleus accumbens. In contrast, the A2AR TM2 peptide significantly reduced the number of A2AR-A2AR homoreceptor complexes in the nucleus accumbens. As to effects on the receptor–receptor interactions in the A2AR-D2R heteroreceptor complexes, the A2AR TM2 peptide did not alter the significant increase in the D2R Ki, high values produced by the A2AR agonist CGS 21680 ex vivo in the ventral striatum. The results indicate that the accumbal A2AR-A2AR homomeric complexes are not involved in mediating the A2AR agonist-induced inhibition of cocaine self-administration.
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Crutzen, François, Marguerite Kreit, and Claude Bragard. "The beet virus Q coat protein readthrough domain is longer than previously reported, with two transmembrane domains." Journal of General Virology 90, no. 3 (2009): 754–58. http://dx.doi.org/10.1099/vir.0.008029-0.
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Ten beet virus Q (BVQ) strains from six different countries were sequenced to characterize the readthrough (RT) domain of the coat protein (CP). The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are FM244643–FM244652. With three nucleotide additions of 5, 285 and 1 nt, the common RT of 76 kDa was found to be longer than the single reference available to date (35 kDa). It is hypothesized that multiple inoculation cycles on Chenopodium quinoa were responsible for these three deletions in the C-terminal part of the BVQ RNA-2 previously described. Two putative transmembrane domains, TM1 and TM2, were predicted in the consensus amino acid sequence of the ten BVQ strains, and the putative BVQ TM2 was aligned with that of potato mop-top virus.
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Jourdan, Alissa D., Scott C. Johnson, and Irene V. Wesley. "Development of a Fluorogenic 5′ Nuclease PCR Assay for Detection of the ail Gene of PathogenicYersinia enterocolitica." Applied and Environmental Microbiology 66, no. 9 (2000): 3750–55. http://dx.doi.org/10.1128/aem.66.9.3750-3755.2000.
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ABSTRACT In this report we describe the development and evaluation of a fluorogenic PCR assay for the detection of pathogenic Yersinia enterocolitica. The assay targets the chromosomally encoded attachment and invasion gene, ail. Three primer-probe sets (TM1, TM2, and TM3) amplifying different, yet overlapping, regions ofail were examined for their specificity and sensitivity. All three primer-probe sets were able to detect between 0.25 and 0.5 pg of purified Y. enterocolitica DNA. TM1 identified all 26Y. enterocolitica strains examined. TM3 was able to detect all strains except one, whereas TM2 was unable to detect 10 of theY. enterocolitica strains tested. None of the primer-probe sets cross-reacted with any of the 21 non-Y. enterocoliticastrains examined. When the TM1 set was utilized, the fluorogenic PCR assay was able to detect ≤4 Y. enterocolitica CFU/ml in pure culture and 10 Y. enterocolitica CFU/ml independent of the presence of 108 CFU of contaminating bacteria per ml. This set was also capable of detecting ≤1 CFU of Y. enterocolitica per g of ground pork or feces after a 24-h enrichment in a Yersinia selective broth.
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Mailler, Sylvain, and François Lott. "Equatorial Mountain Torques and Cold Surge Preconditioning." Journal of the Atmospheric Sciences 67, no. 6 (2010): 2101–20. http://dx.doi.org/10.1175/2010jas3382.1.
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Abstract The evolution of the two components of the equatorial mountain torque (EMT) applied by mountains on the atmosphere is analyzed in the NCEP reanalysis. A strong lagged relationship between the EMT component along the Greenwich axis TM1 and the EMT component along the 90°E axis TM2 is found, with a pronounced signal on TM1 followed by a signal of opposite sign on TM2. It is shown that this result holds for the major massifs (Antarctica, the Tibetan Plateau, the Rockies, and the Andes) if a suitable axis system is used for each of them. For the midlatitude mountains, this relationship is in part associated with the development of cold surges. Following these results, two hypotheses are made: (i) the mountain forcing on the atmosphere is well measured by the regional EMTs and (ii) this forcing partly drives the cold surges. To support these, a purely dynamical linear model is proposed: it is written on the sphere, uses an f-plane quasigeostrophic approximation, and includes the mountain forcings. In this model, a positive (negative) peak in TM1 produced by a mountain massif in the Northern (Southern) Hemisphere is due to a large-scale high surface pressure anomaly poleward of the massif. At a later stage, high pressure and low temperature anomalies develop in the lower troposphere east of the mountain, explaining the signal on TM2 and providing the favorable conditions for the cold surge development. It is concluded that the EMT is a good measure of the dynamical forcing of the atmospheric flow by the mountains and that the poleward forces exerted by mountains on the atmosphere are substantial drivers of the cold surges, at least in their early stage. Therefore, the EMT time series can be an important diagnostic to assess the representation of mountains in general circulation models.
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Takenaga, K., Y. Nakamura, K. Tokunaga, H. Kageyama, and S. Sakiyama. "Isolation and characterization of a cDNA that encodes mouse fibroblast tropomyosin isoform 2." Molecular and Cellular Biology 8, no. 12 (1988): 5561–65. http://dx.doi.org/10.1128/mcb.8.12.5561.
Full textAbstract:
We isolated and characterized a cDNA clone encoding tropomyosin isoform 2 (TM2) from a mouse fibroblast cDNA library. TM2 was found to contain 284 amino acids and was closely related to the rat smooth and skeletal muscle alpha-TMs and the human fibroblast TM3. The amino acid sequence of TM2 showed a nearly complete match with that of human fibroblast TM3 except for the region from amino acids 189 to 213, the sequence of which was identical to those of rat smooth and skeletal muscle alpha-TMs. These results suggest that TM2 is expressed from the same gene that encodes the smooth muscle alpha-TM, the skeletal muscle alpha-TM, and TM3 via an alternative RNA-splicing mechanism. Comparison of the expression of TM2 mRNA in low-metastatic Lewis lung carcinoma P29 cells and high-metastatic D6 cells revealed that it was significantly less in D6 cells than in P29 cells, supporting our previous observations (K. Takenaga, Y. Nakamura, and S. Sakiyama, Mol. Cell. Biol. 8:3934-3937, 1988) at the protein level.
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Takenaga, K., Y. Nakamura, K. Tokunaga, H. Kageyama, and S. Sakiyama. "Isolation and characterization of a cDNA that encodes mouse fibroblast tropomyosin isoform 2." Molecular and Cellular Biology 8, no. 12 (1988): 5561–65. http://dx.doi.org/10.1128/mcb.8.12.5561-5565.1988.
Full textAbstract:
We isolated and characterized a cDNA clone encoding tropomyosin isoform 2 (TM2) from a mouse fibroblast cDNA library. TM2 was found to contain 284 amino acids and was closely related to the rat smooth and skeletal muscle alpha-TMs and the human fibroblast TM3. The amino acid sequence of TM2 showed a nearly complete match with that of human fibroblast TM3 except for the region from amino acids 189 to 213, the sequence of which was identical to those of rat smooth and skeletal muscle alpha-TMs. These results suggest that TM2 is expressed from the same gene that encodes the smooth muscle alpha-TM, the skeletal muscle alpha-TM, and TM3 via an alternative RNA-splicing mechanism. Comparison of the expression of TM2 mRNA in low-metastatic Lewis lung carcinoma P29 cells and high-metastatic D6 cells revealed that it was significantly less in D6 cells than in P29 cells, supporting our previous observations (K. Takenaga, Y. Nakamura, and S. Sakiyama, Mol. Cell. Biol. 8:3934-3937, 1988) at the protein level.
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Zammit, V. A., N. T. Price, F. Fraser, and V. N. Jackson. "Structure-function relationships of the liver and muscle isoforms of carnitine palmitoyltransferase I." Biochemical Society Transactions 29, no. 2 (2001): 287–91. http://dx.doi.org/10.1042/bst0290287.
Full textAbstract:
Elucidation of the membrane topology of carnitine palmitoyltransferase (CPT) I showed that the extreme N-terminus is involved in determining the sensitivity of the liver (L) isoform to malonyl-CoA and suggested that interaction between the two cytosolic segments of the CPT I molecule determines the kinetic characteristics of the enzyme. Work with chimaeric liver/muscle-isoform (L/M) proteins constructed from all six possible combinations of three domains [N-terminus plus transmembrane domain 1 (TM1), loop plus TM2 and C-domain] expressed in Pichia pastoris showed that the precise N-C and TM1-TM2 pairings determine the overall kinetic parameters of the protein. Discrete short sequences within the respective N-terminal regions have negative or positive effects on malonyl-CoA sensitivity (L-isoform) or the Km for carnitine (M-isoform) in the full-length proteins, thus imparting to them their distinctive kinetic characteristics. Interactions within N-terminal domains also seem to be important in the targeting of the protein to microsomes in the P. pastoris expression system.
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Ye, Libin, Xiaoyun Su, George E. Schmitz, et al. "Molecular and Biochemical Analyses of the GH44 Module of CbMan5B/Cel44A, a Bifunctional Enzyme from the Hyperthermophilic Bacterium Caldicellulosiruptor bescii." Applied and Environmental Microbiology 78, no. 19 (2012): 7048–59. http://dx.doi.org/10.1128/aem.02009-12.
Full textAbstract:
ABSTRACTA large polypeptide encoded in the genome of the thermophilic bacteriumCaldicellulosiruptor besciiwas determined to consist of two glycoside hydrolase (GH) modules separated by two carbohydrate-binding modules (CBMs). Based on the detection of mannanase and endoglucanase activities in the N-terminal GH5 and the C-terminal GH44 module, respectively, the protein was designated CbMan5B/Cel44A. A GH5 module with >99% identity from the same organism was characterized previously (X. Su, R. I. Mackie, and I. K. Cann, Appl. Environ. Microbiol.78:2230-2240, 2012); therefore, attention was focused on CbMan5A/Cel44A-TM2 (or TM2), which harbors the GH44 module and the two CBMs. On cellulosic substrates, TM2 had an optimal temperature and pH of 85°C and 5.0, respectively. Although the amino acid sequence of the GH44 module of TM2 was similar to those of other GH44 modules that hydrolyzed cello-oligosaccharides, cellulose, lichenan, and xyloglucan, it was unique that TM2 also displayed modest activity on mannose-configured substrates and xylan. The TM2 protein also degraded Avicel with higher specific activity than activities reported for its homologs. The GH44 catalytic module is composed of a TIM-like domain and a β-sandwich domain, which consists of one β-sheet at the N terminus and nine β-sheets at the C terminus. Deletion of one or more β-sheets from the β-sandwich domain resulted in insoluble proteins, suggesting that the β-sandwich domain is essential for proper folding of the polypeptide. Combining TM2 with three other endoglucanases fromC. besciiled to modest synergistic activities during degradation of cellulose, and based on our results, we propose a model for cellulose hydrolysis and utilization byC. bescii.
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SALDANHA, DEJANIRA LUDERITZ, MARIA DO CARMO LIMA E. CUNHA DO CARMO LIMA E. CUNHA, and JOSÉ CARUSO MORESCO DANNI. "Identificação de Rochas Ultramáficas por Imagens Digitais TM - Landsat 5 no Escudo Sul-rio-grandense, RS." Pesquisas em Geociências 35, no. 1 (2008): 21. http://dx.doi.org/10.22456/1807-9806.17892.
Full textAbstract:
Principal Components of Landsat TM images were used aiming to identify occurrences of ultramafic rocks in Rio Grande do Sul State, Brazil. Principal Component involving the pairs of bands TM1-TM5, TM4-TM2 and TM5-TM7 showed to be the best for the purposes of the study. Field surveys confirmed the results obtained by digital processing of the images.
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Bond, Ashley D., Michael D. Burkitt, David Sawbridge, Bernard M. Corfe, and Chris S. Probert. "Correlation between Faecal Tumour M2 Pyruvate Kinase and Colonoscopy for the Detection of Adenomatous Neoplasia in a Secondary Care Cohort." Journal of Gastrointestinal and Liver Diseases 25, no. 1 (2016): 71–77. http://dx.doi.org/10.15403/jgld.2014.1121.251.m2p.
Full textAbstract:
Background & Aims: Colorectal cancer screening programmes that target detection and excision of adenomatous colonic polyps have been shown to reduce colorectal cancer related mortality. Many screening programmes include an initial faecal occult blood test (FOBt) prior to colonoscopy. To refine the selection of patients for colonoscopy other faecal-based diagnostic tools have been proposed, including tumour M2-pyruvate kinase (tM2-PK). To determine whether tM2-PK quantification may have a role in diverse settings we have assessed the assay in a cohort of patients derived from both the England bowel cancer screening programme (BCSP) and symptomatic individuals presenting to secondary care.
 Method. Patients undergoing colonoscopy provided faecal samples prior to bowel preparation. Faecal tM2-PK concentrations were measured by ELISA. Sensitivity, specificity, positive predictive value, negative predictive value and ROC analyses were calculated.
 Results. Ninety-six patients returned faecal samples: 50 of these with adenomas and 7 with cancer. Median age was 68. Median faecal tM2-PK concentration was 3.8 U/mL for individuals without neoplastic findings at colonoscopy, 7.7 U/mL in those with adenomas and 24.4 U/mL in subjects with colorectal cancer (both, p=0.01). ROC analysis demonstrated an AUROC of 0.66 (sensitivity 72.4%, specificity 48.7%, positive predictive value 67.7%, negative predictive value 36.7%). Amongst BCSP patients with a prior positive FOBt faecal tM2-PK was more abundant (median 6.4 U/mL, p=0.03) and its diagnostic accuracy was greater (AUROC 0.82).
 Conclusion. Our findings confirm that faecal tM2-PK ELISA may have utility as an adjunct to FOBt in a screening context, but do not support its use in symptomatic patients.
 Abbreviations: BCSP: Bowel cancer screening programme; EMR: Endoscopic mucosal resection; FAP: Familial adenomatous polyposis; FOBt: Faecal occult blood testing; NHS: National Health Service; tM2-PK: tumour M2-pyruvate kinase.
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ZHANG, Jian-Ting. "Determinant of the extracellular location of the N-terminus of human multidrug-resistance-associated protein." Biochemical Journal 348, no. 3 (2000): 597–606. http://dx.doi.org/10.1042/bj3480597.
Full textAbstract:
Multidrug-resistance-associated protein (MRP) is a member of the ATP-binding cassette (ABC) membrane-transport superfamily and is responsible for multidrug resistance in cancer cells. Distinct from other members of the ABC superfamily, MRP has three membrane-spanning domains (MSDs) and the N-terminus is located extracellularly. It has been shown that the first MSD (MSD1) with an extracellular N-terminus is important for MRP function. To address what ensures the generation of this structural organization of MRP and to understand in general the molecular mechanism of membrane folding of polytopic proteins with extracellular N-termini, the biogenesis of MSD1 in human MRP1 was examined using an in vitro expression system. Surprisingly, the second transmembrane segment (TM2) in MSD1 was found to play a critical role in the correct membrane translocation and folding of MSD1 in human MRP1. TM2 not only plays an essential role to ensure the N-terminus-outside/C-terminus-inside orientation of TM1 with an extracellular N-terminus, it can also translocate into membranes post-translationally in a signal-recognition particle and ribosome-dependent manner to provide an additional insurance for correct folding of MSD1 in MRP. These findings suggest that TM2 in a polytopic membrane protein with an extracellular N-terminus may play a critical role in controlling correct membrane translocation and folding of the protein in general.
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Takenaga, K., Y. Nakamura, and S. Sakiyama. "Differential expression of a tropomyosin isoform in low- and high-metastatic Lewis lung carcinoma cells." Molecular and Cellular Biology 8, no. 9 (1988): 3934–37. http://dx.doi.org/10.1128/mcb.8.9.3934.
Full textAbstract:
Two-dimensional electrophoretograms of newly synthesized polypeptides from low-metastatic (P29) and high-metastatic (D6) Lewis lung carcinoma cells were compared. The results showed that the synthesis of tropomyosin 2 (TM2) was significantly less in D6 cells than in P29 cells. Furthermore, suppression of TM2 synthesis was induced in P29 cells during incubation in medium containing dimethyl sulfoxide or butyric acid, which induced the metastatic phenotype of P29 cells. These results suggest that the suppression of TM2 synthesis is linked to the metastatic potential of Lewis lung carcinoma cells.
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Takenaga, K., Y. Nakamura, and S. Sakiyama. "Differential expression of a tropomyosin isoform in low- and high-metastatic Lewis lung carcinoma cells." Molecular and Cellular Biology 8, no. 9 (1988): 3934–37. http://dx.doi.org/10.1128/mcb.8.9.3934-3937.1988.
Full textAbstract:
Two-dimensional electrophoretograms of newly synthesized polypeptides from low-metastatic (P29) and high-metastatic (D6) Lewis lung carcinoma cells were compared. The results showed that the synthesis of tropomyosin 2 (TM2) was significantly less in D6 cells than in P29 cells. Furthermore, suppression of TM2 synthesis was induced in P29 cells during incubation in medium containing dimethyl sulfoxide or butyric acid, which induced the metastatic phenotype of P29 cells. These results suggest that the suppression of TM2 synthesis is linked to the metastatic potential of Lewis lung carcinoma cells.
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Gallant, Cynthia, Sarah Appel, Philip Graceffa, et al. "Tropomyosin variants describe distinct functional subcellular domains in differentiated vascular smooth muscle cells." American Journal of Physiology-Cell Physiology 300, no. 6 (2011): C1356—C1365. http://dx.doi.org/10.1152/ajpcell.00450.2010.
Full textAbstract:
Tropomyosin (Tm) is known to be an important gatekeeper of actin function. Tm isoforms are encoded by four genes, and each gene produces several variants by alternative splicing, which have been proposed to play roles in motility, proliferation, and apoptosis. Smooth muscle studies have focused on gizzard smooth muscle, where a heterodimer of Tm from the α-gene (Tmsm-α) and from the β-gene (Tmsm-β) is associated with contractile filaments. In this study we examined Tm in differentiated mammalian vascular smooth muscle (dVSM). Liquid chromatography-tandem mass spectrometry (LC MS/MS) analysis and Western blot screening with variant-specific antibodies revealed that at least five different Tm proteins are expressed in this tissue: Tm6 (Tmsm-α) and Tm2 from the α-gene, Tm1 (Tmsm-β) from the β-gene, Tm5NM1 from the γ-gene, and Tm4 from the δ-gene. Tm6 is by far most abundant in dVSM followed by Tm1, Tm2, Tm5NM1, and Tm4. Coimmunoprecipitation and coimmunofluorescence studies demonstrate that Tm1 and Tm6 coassociate with different actin isoforms and display different intracellular localizations. Using an antibody specific for cytoplasmic γ-actin, we report here the presence of a γ-actin cortical cytoskeleton in dVSM cells. Tm1 colocalizes with cortical cytoplasmic γ-actin and coprecipitates with γ-actin. Tm6, on the other hand, is located on contractile bundles. These data indicate that Tm1 and Tm6 do not form a classical heterodimer in dVSM but rather describe different functional cellular compartments.
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Waelbroeck, M., J. Perret, P. Vertongen, M. Van Craenenbroeck, and P. Robberecht. "Identification of secretin, vasoactive intestinal peptide and glucagon binding sites: from chimaeric receptors to point mutations." Biochemical Society Transactions 30, no. 4 (2002): 437–41. http://dx.doi.org/10.1042/bst0300437.
Full textAbstract:
We have identified two basic residues that are important for the recognition of secretin and vasoactive intestinal peptide (VIP) by their respective receptors. These two peptides containing an Asp residue at position 3 interacted with an arginine residue in transmembrane helix 2 (TM2) of the receptor, and the lysine residue in extracellular loop 1 (ECL1) stabilized the active receptor conformation induced by the ligand. The glucagon receptor possesses a Lys instead of an Arg in TM2, and an Ile instead of Lys in ECL1; it markedly prefers a Gln side chain in position 3 of the ligand. Our results suggested that, in the wild-type receptor, the Ile side chain prevented access to the TM2 Lys side chain, but oriented the glucagon Gln3 side chain to its proper binding site. In the double mutant, the ECL1 Lys allowed an interaction between negatively charged residues in position 3 of glucagon and the TM2 Arg, resulting in efficient receptor activation by [Asp3]glucagon as well as by glucagon.
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Cheung, Joanne C., Jing Li, and Reinhart A. F. Reithmeier. "Topology of transmembrane segments 1–4 in the human chloride/bicarbonate anion exchanger 1 (AE1) by scanning N-glycosylation mutagenesis." Biochemical Journal 390, no. 1 (2005): 137–44. http://dx.doi.org/10.1042/bj20050315.
Full textAbstract:
Human AE1 (anion exchanger 1), or Band 3, is an abundant membrane glycoprotein found in the plasma membrane of erythrocytes. The physiological role of the protein is to carry out chloride/bicarbonate exchange across the plasma membrane, a process that increases the carbon-dioxide-carrying capacity of blood. To study the topology of TMs (transmembrane segments) 1–4, a series of scanning N-glycosylation mutants were created spanning the region from EC (extracellular loop) 1 to EC2 in full-length AE1. These constructs were expressed in HEK-293 (human embryonic kidney) cells, and their N-glycosylation efficiencies were determined. Unexpectedly, positions within putative TMs 2 and 3 could be efficiently glycosylated. In contrast, the same positions were very poorly glycosylated when present in mutant AE1 with the SAO (Southeast Asian ovalocytosis) deletion (ΔA400–A408) in TM1. These results suggest that the TM2–3 region of AE1 may become transiently exposed to the endoplasmic reticulum lumen during biosynthesis, and that there is a competition between proper folding of the region into the membrane and N-glycosylation at introduced sites. The SAO deletion disrupts the proper integration of TMs 1–2, probably leaving the region exposed to the cytosol. As a result, engineered N-glycosylation acceptor sites in TM2–3 could not be utilized by the oligosaccharyltransferase in this mutant form of AE1. The properties of TM2–3 suggest that these segments form a re-entrant loop in human AE1.
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Young, Mark T., Yi-Hong Zhang, Lishuang Cao, Helen Broomhead, and Lin-Hua Jiang. "Role of the domain encompassing Arg304–Ile328 in rat P2X2 receptor conformation revealed by alterations in complex glycosylation at Asn298." Biochemical Journal 416, no. 1 (2008): 137–43. http://dx.doi.org/10.1042/bj20081182.
Full textAbstract:
The final 25 amino acids of the ectodomain of the P2X receptors, immediately prior to the second TM (transmembrane domain) (pre-TM2: Arg304–Ile328 in rat P2X2), are highly conserved. Whole-cell patch clamp recordings showed that single cysteine substitutions in the N-terminal half of pre-TM2 (Arg304–Ile314) led to loss of function at Arg304, Leu306, Lys308 and Ile312. Cysteine substitutions within this region also resulted in a significant reduction in the apparent molecular mass of receptors, due to loss of complex glycosylation at the nearby acceptor site Asn298, which was not seen for the C-terminal portion of pre-TM2 (Asp315–Ile328). The reduction in complex glycosylation was not due to reduced cell-surface presentation, demonstrating that glycosylation at Asn298 was acting as a sensor of subtle changes in receptor conformation within the pre-TM2 region. When this N-glycan site was repositioned closer to the plasma membrane by mutagenesis (N298S together with G299N, T300N, T301N or T303N), glycosylation was restored at G299N and T300N, but was impaired for T301N and completely absent for T303N. These results suggest that the region in the vicinity of Asp315 is at the plasma membrane interface and that the N-terminal portion of pre-TM2 (Arg304–Ile314) is important for the correct conformation of the receptor at the extracellular face of the membrane.
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Chang, Hao, Philip M. Smallwood, John Williams, and Jeremy Nathans. "Intramembrane Proteolysis of Astrotactins." Journal of Biological Chemistry 292, no. 8 (2017): 3506–16. http://dx.doi.org/10.1074/jbc.m116.768077.
Full textAbstract:
Astrotactins are vertebrate-specific membrane proteins implicated in neuron-glia interactions during central nervous system development and in hair follicle polarity during skin development. By studying epitope-tagged derivatives of mouse astrotactin-2 (Astn2) produced in transfected cells, we determined that the amino and carboxyl termini reside in the extracellular space and are initially linked by two transmembrane segments and a single cytoplasmic domain. We further show that Astn2 undergoes proteolytic cleavage in the second transmembrane domain (TM2) and that a disulfide bond holds the resulting two fragments together. Recombinant Astn1 also undergoes TM2 cleavage, as does Astn2 isolated from mouse cerebellum. Astn2 intramembrane proteolysis is insensitive to replacement of TM2 by the transmembrane domain of CD74 or by 21 alanines. However, replacement of TM2 by the transmembrane domain of CD4, the asialoglycoprotein receptor, or the transferrin receptor eliminates intramembrane proteolysis, as does leucine substitution of residues that overlap or are immediately upstream of the cleavage site. Replacement of the transmembrane domain of CD74 or the asialoglycoprotein receptor with Astn2 TM2 leads to the appearance of a carboxyl-terminal fragment consistent with intramembrane proteolysis. These experiments define a highly unusual transmembrane topology for the astrotactins, reveal intramembrane proteolysis as a feature of astrotactin maturation, and constrain the substrate sequences that are permissive for cleavage of one type 2 transmembrane segment.
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Meacock, Suzanna L., Fabienne J. L. Lecomte, Samuel G. Crawshaw, and Stephen High. "Different Transmembrane Domains Associate with Distinct Endoplasmic Reticulum Components during Membrane Integration of a Polytopic Protein." Molecular Biology of the Cell 13, no. 12 (2002): 4114–29. http://dx.doi.org/10.1091/mbc.e02-04-0198.
Full textAbstract:
We have been studying the insertion of the seven transmembrane domain (TM) protein opsin to gain insights into how the multiple TMs of polytopic proteins are integrated at the endoplasmic reticulum (ER). We find that the ER components associated with the first and second TMs of the nascent opsin polypeptide chain are clearly distinct. The first TM (TM1) is adjacent to the α and β subunits of the Sec61 complex, and a novel component, a protein associated with the ER translocon of 10 kDa (PAT-10). The most striking characteristic of PAT-10 is that it remains adjacent to TM1 throughout the biogenesis and membrane integration of the full-length opsin polypeptide. TM2 is also found to be adjacent to Sec61α and Sec61β during its membrane integration. However, TM2 does not form any adducts with PAT-10; rather, a transient association with the TRAM protein is observed. We show that the association of PAT-10 with opsin TM1 does not require theN-glycosylation of the nascent chain and occurs irrespective of the amino acid sequence and transmembrane topology of TM1. We conclude that the precise makeup of the ER membrane insertion site can be distinct for the different transmembrane domains of a polytopic protein. We find that the environment of a particular TM can be influenced by both the “stage” of nascent chain biosynthesis reached, and the TM's relative location within the polypeptide.
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Siegler, Vera D., and Volker Bruss. "Role of Transmembrane Domains of Hepatitis B Virus Small Surface Proteins in Subviral-Particle Biogenesis." Journal of Virology 87, no. 3 (2012): 1491–96. http://dx.doi.org/10.1128/jvi.02500-12.
Full textAbstract:
ABSTRACTThe hepatitis B virus (HBV) surface proteins not only are incorporated into the virion envelope but in addition form subviral particles (SVP) consisting solely of surface proteins and lipids. Heterologous expression of the small HBV envelope protein S produces secreted spherical SVP 20 nm in diameter, with approximately 100 S molecules per particle. The pathway leading from the initial S translation product as a multispanning transmembrane protein to the final SVP is largely unknown. To investigate the role of the four transmembrane domains (TM) of S in this process, we introduced mutations in these regions and characterized their effects on SVP formation in transfected Huh7 cells. We found that the insertion of one amino acid in the center of the α-helix of TM1 or the exchange of TM1 with a heterologous TM blocked SVP release and SVP formation by coexpressed wild-type S chains in a transdominant negative fashion. Surprisingly, this effect was partially neutralized when the mutations were expressed in the background of the HBV surface protein M, suggesting that mutations in TM1 could partially be complemented by the pre-S2 domain. The exchange of TM2 with heterologous TMs that form α-helices of the same lengths was also incompatible with SVP formation. However, these mutants no longer blocked SVP formation by coexpressed wild-type S. We conclude that TM2 is essential for the stable assembly of S chains by establishing intramembrane interactions.
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Valdez Capuccino, Juan M., Payal Chatterjee, Isaac E. García, et al. "The connexin26 human mutation N14K disrupts cytosolic intersubunit interactions and promotes channel opening." Journal of General Physiology 151, no. 3 (2018): 328–41. http://dx.doi.org/10.1085/jgp.201812219.
Full textAbstract:
A group of human mutations within the N-terminal (NT) domain of connexin 26 (Cx26) hemichannels produce aberrant channel activity, which gives rise to deafness and skin disorders, including keratitis-ichthyosis-deafness (KID) syndrome. Structural and functional studies indicate that the NT of connexin hemichannels is folded into the pore, where it plays important roles in permeability and gating. In this study, we explore the molecular basis by which N14K, an NT KID mutant, promotes gain of function. In macroscopic and single-channel recordings, we find that the N14K mutant favors the open conformation of hemichannels, shifts calcium and voltage sensitivity, and slows deactivation kinetics. Multiple copies of MD simulations of WT and N14K hemichannels, followed by the Kolmogorov–Smirnov significance test (KS test) of the distributions of interaction energies, reveal that the N14K mutation significantly disrupts pairwise interactions that occur in WT hemichannels between residue K15 of one subunit and residue E101 of the adjacent subunit (E101 being located at the transition between transmembrane segment 2 [TM2] and the cytoplasmic loop [CL]). Double mutant cycle analysis supports coupling between the NT and the TM2/CL transition in WT hemichannels, which is disrupted in N14K mutant hemichannels. KS tests of the α carbon correlation coefficients calculated over MD trajectories suggest that the effects of the N14K mutation are not confined to the K15–E101 pairs but extend to essentially all pairwise residue correlations between the NT and TM2/CL interface. Together, our data indicate that the N14K mutation increases hemichannel open probability by disrupting interactions between the NT and the TM2/CL region of the adjacent connexin subunit. This suggests that NT–TM2/CL interactions facilitate Cx26 hemichannel closure.
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Ohto, H., H. Maeda, Y. Shibata, et al. "A novel leukocyte differentiation antigen: two monoclonal antibodies TM2 and TM3 define a 120-kd molecule present on neutrophils, monocytes, platelets, and activated lymphoblasts." Blood 66, no. 4 (1985): 873–81. http://dx.doi.org/10.1182/blood.v66.4.873.873.
Full textAbstract:
Abstract We produced two hybridomas by fusion of mouse myeloma cells with splenocytes from a mouse immunized with the THP-1 human monocytoid leukemia cell line. Two cloned hybridoma cell lines, designated as TM2 and TM3, were obtained. They secreted antibodies against a unique cell surface antigen expressed on all normal peripheral blood monocytes, neutrophilic granulocytes, platelets, and mitogen-induced lymphoblasts, some cells from patients with immature-type lymphoid leukemias. However, the antibodies reacted neither with large numbers of peripheral blood lymphocytes nor with red cells. Cross-blocking studies showed that these monoclonal antibodies recognized the same or a nearly positioned antigen epitope. Immunoprecipitation of THP-1 cell extract with TM2 or TM3 under reducing and nonreducing conditions yielded a specific band of mol wt equal to 120,000 daltons. This determinant appeared to be involved in granulocyte chemotaxis, since neutrophilic granulocytes exposed to TM2 or TM3 showed a significant decrease in chemotaxis toward endotoxin-activated serum. These two monoclonal antibodies did not affect O2- release or luminol-dependent chemiluminescence of neutrophils. Moreover, they did not alter platelet aggregation induced by thrombin. TM2 and TM3 will provide a new reagent in defining the linkage between lymphoid and myeloid differentiation and intermyeloid development.
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Ohto, H., H. Maeda, Y. Shibata, et al. "A novel leukocyte differentiation antigen: two monoclonal antibodies TM2 and TM3 define a 120-kd molecule present on neutrophils, monocytes, platelets, and activated lymphoblasts." Blood 66, no. 4 (1985): 873–81. http://dx.doi.org/10.1182/blood.v66.4.873.bloodjournal664873.
Full textAbstract:
We produced two hybridomas by fusion of mouse myeloma cells with splenocytes from a mouse immunized with the THP-1 human monocytoid leukemia cell line. Two cloned hybridoma cell lines, designated as TM2 and TM3, were obtained. They secreted antibodies against a unique cell surface antigen expressed on all normal peripheral blood monocytes, neutrophilic granulocytes, platelets, and mitogen-induced lymphoblasts, some cells from patients with immature-type lymphoid leukemias. However, the antibodies reacted neither with large numbers of peripheral blood lymphocytes nor with red cells. Cross-blocking studies showed that these monoclonal antibodies recognized the same or a nearly positioned antigen epitope. Immunoprecipitation of THP-1 cell extract with TM2 or TM3 under reducing and nonreducing conditions yielded a specific band of mol wt equal to 120,000 daltons. This determinant appeared to be involved in granulocyte chemotaxis, since neutrophilic granulocytes exposed to TM2 or TM3 showed a significant decrease in chemotaxis toward endotoxin-activated serum. These two monoclonal antibodies did not affect O2- release or luminol-dependent chemiluminescence of neutrophils. Moreover, they did not alter platelet aggregation induced by thrombin. TM2 and TM3 will provide a new reagent in defining the linkage between lymphoid and myeloid differentiation and intermyeloid development.
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Migdał-Mikuli, Anna, and Elżbieta Szostak. "Phase Polymorphism of [Ni(DMSO)6](ClO4)2 Studied by Differential Scanning Calorimetry." Zeitschrift für Naturforschung A 62, no. 1-2 (2007): 67–74. http://dx.doi.org/10.1515/zna-2007-1-210.
Full textAbstract:
Six solid phases of [Ni(DMSO)6](ClO4)2 have been detected by differential scanning calorimetry (DSC). The five phase transitions were detected between the following solid phases: metastable KIII ↔ undercooled K0 at TC5 = 326 K, stable KIb → stable KIa at TC4 = 350 K, metastable KII ↔ undercooled KI at TC3 = 353 K, stable KIa → stable KI at TC2 = 365 K and stable KI → stable K0 at TC1 = 380 K. At Tm2 = 459 K the title compound partially dissolves in DMSO, which arises from the decomposition of [Ni(DMSO)6](ClO4)2 to [Ni(DMSO)5](ClO4)2, and at Tm1 = 526 K created in this way a substance which completely melts. From the entropy changes at the melting point and at phase transitions it can be concluded that the phases K0 and undercooled K0 are orientationally dynamically disordered crystals. The stable phases KI, KIa, KIb and the metastable phases KII and KIII are more or less ordered solids.
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Yu, Rui Hong, Ting Xi Liu, and Rui Ying Hao. "Water Depth Retrieval Model by Remote Sensing in Wuliangsuhai Wetland." Advanced Materials Research 726-731 (August 2013): 4674–77. http://dx.doi.org/10.4028/www.scientific.net/amr.726-731.4674.
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Wuliangsuhai wetland located in Inner Mongolia Autonomous Region (IMAR) is selected as the study area to analyze the wetland environment evolution. Wuliangsuhai wetland is the largest freshwater lake in the Yellow River basin, and also the largest natural wetland along the same latitude on the earth. Through the optimization comparison of multiple wave-band reflection combination, the regression equation of the water depth and wave band reflection combination of TM3, TM2 and TM1 is established based on the remote sensing principles.
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Allsopp, Rebecca C., Louise K. Farmer, Alistair G. Fryatt, and Richard J. Evans. "P2X Receptor Chimeras Highlight Roles of the Amino Terminus to Partial Agonist Efficacy, the Carboxyl Terminus to Recovery from Desensitization, and Independent Regulation of Channel Transitions." Journal of Biological Chemistry 288, no. 29 (2013): 21412–21. http://dx.doi.org/10.1074/jbc.m113.464651.
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P2X receptor subtypes can be distinguished by their sensitivity to ATP analogues and selective antagonists. We have used chimeras between human P2X1 and P2X2 receptors to address the contribution of the extracellular ligand binding loop, transmembrane segments (TM1 and TM2), and intracellular amino and carboxyl termini to the action of partial agonists (higher potency and efficacy of BzATP and Ap5A at P2X1 receptors) and antagonists. Sensitivity to the antagonists NF449, suramin, and PPADS was conferred by the nature of the extracellular loop (e.g. nanomolar for NF449 at P2X1 and P2X2-1EXT and micromolar at P2X2 and P2X1-2EXT). In contrast, the effectiveness of partial agonists was similar to P2X1 levels for both of the loop transfers, suggesting that interactions with the rest of the receptor played an important role. Swapping TM2 had reciprocal effects on partial agonist efficacy. However, TM1 swaps increased partial agonist efficacy at both chimeras, and this was similar for swaps of both TM1 and 2. Changing the amino terminus had no effect on agonist potency but increased partial agonist efficacy at P2X2-1N and decreased it at P2X1-2N chimeras, demonstrating that potency and efficacy can be independently regulated. Chimeras and point mutations also identified residues in the carboxyl terminus that regulated recovery from channel desensitization. These results show that interactions among the intracellular, transmembrane, and extracellular portions of the receptor regulate channel properties and suggest that transitions to channel opening, the behavior of the open channel, and recovery from the desensitized state can be controlled independently.
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Witan, Julian, Christian Monzel, Patrick D. Scheu, and Gottfried Unden. "The sensor kinase DcuS of Escherichia coli: two stimulus input sites and a merged signal pathway in the DctA/DcuS sensor unit." Biological Chemistry 393, no. 11 (2012): 1291–97. http://dx.doi.org/10.1515/hsz-2012-0229.
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Abstract The membrane-integral sensor kinase DcuS of Escherichia coli consists of a periplasmically located sensory PASP domain, transmembrane helices TM1 and TM2, a cytoplasmic PASC domain and the kinase domain. Stimulus (C4-dicarboxylate) binding at PASP is required to stimulate phosphorylation of the kinase domain, resulting in phosphoryl transfer to the response regulator DcuR. PASC functions as a signaling device or a relay in signal transfer from TM2 to the kinase. Phosphorylated DcuR induces the expression of the target genes. Sensing by DcuS requires the presence of the C4-dicarboxylate transporter DctA during aerobic growth. DctA forms a sensor unit with DcuS, and a short C-terminal sequence of DctA forming the putative helix 8b is required for interaction with DcuS. Helix 8b contains a LDXXXLXXXL motif that is essential for function and interaction. DcuS requires the PASC domain for signal perception from DctA. Thus, DcuS and DctA form a DctA/DcuS sensory unit, and DcuS perceives stimuli from two different sites (PASP and DctA). The signal transfer pathways are supposed to merge at PASC. The fumarate/succinate antiporter DcuB takes over the role as a co-sensor of DcuS under anaerobic growth conditions.
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Filippova, Natalia, Virginia E. Wotring та David S. Weiss. "Evidence that the TM1-TM2 Loop Contributes to the ρ1 GABA Receptor Pore". Journal of Biological Chemistry 279, № 20 (2004): 20906–14. http://dx.doi.org/10.1074/jbc.m401012200.
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Merkx, Evert P. J., Maarten P. Plokker, and Erik van der Kolk. "The potential of transparent sputtered NaI:Tm2+, CaBr2:Tm2+, and CaI2:Tm2+ thin films as luminescent solar concentrators." Solar Energy Materials and Solar Cells 223 (May 2021): 110944. http://dx.doi.org/10.1016/j.solmat.2020.110944.
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Liu, Guoxia, Sergey I. Zakharov, Yongneng Yao, Steven O. Marx та Arthur Karlin. "Positions of the cytoplasmic end of BK α S0 helix relative to S1–S6 and of β1 TM1 and TM2 relative to S0–S6". Journal of General Physiology 145, № 3 (2015): 185–99. http://dx.doi.org/10.1085/jgp.201411337.
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The large-conductance, voltage- and Ca2+-gated K+ (BK) channel consists of four α subunits, which form a voltage- and Ca2+-gated channel, and up to four modulatory β subunits. The β1 subunit is expressed in smooth muscle, where it slows BK channel kinetics and shifts the conductance–voltage (G-V) curve to the left at [Ca2+] > 2 µM. In addition to the six transmembrane (TM) helices, S1–S6, conserved in all voltage-dependent K+ channels, BK α has a unique seventh TM helix, S0, which may contribute to the unusual rightward shift in the G-V curve of BK α in the absence of β1 and to a leftward shift in its presence. Such a role is supported by the close proximity of S0 to S3 and S4 in the voltage-sensing domain. Furthermore, on the extracellular side of the membrane, one of the two TM helices of β1, TM2, is adjacent to S0. We have now analyzed induced disulfide bond formation between substituted Cys residues on the cytoplasmic side of the membrane. There, in contrast, S0 is closest to the S2–S3 loop, from which position it is displaced on the addition of β1. The cytoplasmic ends of β1 TM1 and TM2 are adjacent and are located between the S2–S3 loop of one α subunit and S1 of a neighboring α subunit and are not adjacent to S0; i.e., S0 and TM2 have different trajectories through the membrane. In the absence of β1, 70% of disulfide bonding of W43C (S0) and L175C (S2–S3) has no effect on V50 for activation, implying that the cytoplasmic end of S0 and the S2–S3 loop move in concert, if at all, during activation. Otherwise, linking them together in one state would obstruct the transition to the other state, which would certainly change V50.
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Yeung, Priscilla S. W., Megumi Yamashita, Christopher E. Ing, Régis Pomès, Douglas M. Freymann, and Murali Prakriya. "Mapping the functional anatomy of Orai1 transmembrane domains for CRAC channel gating." Proceedings of the National Academy of Sciences 115, no. 22 (2018): E5193—E5202. http://dx.doi.org/10.1073/pnas.1718373115.
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Store-operated Orai1 channels are activated through a unique inside-out mechanism involving binding of the endoplasmic reticulum Ca2+ sensor STIM1 to cytoplasmic sites on Orai1. Although atomic-level details of Orai structure, including the pore and putative ligand binding domains, are resolved, how the gating signal is communicated to the pore and opens the gate is unknown. To address this issue, we used scanning mutagenesis to identify 15 residues in transmembrane domains (TMs) 1–4 whose perturbation activates Orai1 channels independently of STIM1. Cysteine accessibility analysis and molecular-dynamics simulations indicated that constitutive activation of the most robust variant, H134S, arises from a pore conformational change that opens a hydrophobic gate to augment pore hydration, similar to gating evoked by STIM1. Mutational analysis of this locus suggests that H134 acts as steric brake to stabilize the closed state of the channel. In addition, atomic packing analysis revealed distinct functional contacts between the TM1 pore helix and the surrounding TM2/3 helices, including one set mediated by a cluster of interdigitating hydrophobic residues and another by alternative ridges of polar and hydrophobic residues. Perturbing these contacts via mutagenesis destabilizes STIM1-mediated Orai1 channel gating, indicating that these bridges between TM1 and the surrounding TM2/3 ring are critical for conveying the gating signal to the pore. These findings help develop a framework for understanding the global conformational changes and allosteric interactions between topologically distinct domains that are essential for activation of Orai1 channels.
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Han, Yejun, Dylan Dodd, Charles W. Hespen та ін. "Comparative Analyses of Two Thermophilic Enzymes Exhibiting both β-1,4 Mannosidic and β-1,4 Glucosidic Cleavage Activities from Caldanaerobius polysaccharolyticus". Journal of Bacteriology 192, № 16 (2010): 4111–21. http://dx.doi.org/10.1128/jb.00257-10.
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ABSTRACT The hydrolysis of polysaccharides containing mannan requires endo-1,4-β-mannanase and 1,4-β-mannosidase activities. In the current report, the biochemical properties of two endo-β-1,4-mannanases (Man5A and Man5B) from Caldanaerobius polysaccharolyticus were studied. Man5A is composed of an N-terminal signal peptide (SP), a catalytic domain, two carbohydrate-binding modules (CBMs), and three surface layer homology (SLH) repeats, whereas Man5B lacks the SP, CBMs, and SLH repeats. To gain insights into how the two glycoside hydrolase family 5 (GH5) enzymes may aid the bacterium in energy acquisition and also the potential application of the two enzymes in the biofuel industry, two derivatives of Man5A (Man5A-TM1 [TM1 stands for truncational mutant 1], which lacks the SP and SLH repeats, and Man5A-TM2, which lacks the SP, CBMs, and SLH repeats) and the wild-type Man5B were biochemically analyzed. The Man5A derivatives displayed endo-1,4-β-mannanase and endo-1,4-β-glucanase activities and hydrolyzed oligosaccharides with a degree of polymerization (DP) of 4 or higher. Man5B exhibited endo-1,4-β-mannanase activity and little endo-1,4-β-glucanase activity; however, this enzyme also exhibited 1,4-β-mannosidase and cellodextrinase activities. Man5A-TM1, compared to either Man5A-TM2 or Man5B, had higher catalytic activity with soluble and insoluble polysaccharides, indicating that the CBMs enhance catalysis of Man5A. Furthermore, Man5A-TM1 acted synergistically with Man5B in the hydrolysis of β-mannan and carboxymethyl cellulose. The versatility of the two enzymes, therefore, makes them a resource for depolymerization of mannan-containing polysaccharides in the biofuel industry. Furthermore, on the basis of the biochemical and genomic data, a molecular mechanism for utilization of mannan-containing nutrients by C. polysaccharolyticus is proposed.
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Nishikawa, Koichi, та Neil L. Harrison. "The Actions of Sevoflurane and Desflurane on the γ-Aminobutyric Acid Receptor Type A". Anesthesiology 99, № 3 (2003): 678–84. http://dx.doi.org/10.1097/00000542-200309000-00024.
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Background Previous studies have shown that specific amino acid residues in the putative second transmembrane segment (TM2) of the gamma-aminobutyric acid receptor type A (GABAA) receptor play a critical role in the enhancement of GABAA receptor function by halothane, enflurane, and isoflurane. However, very little is known about the actions of sevoflurane and desflurane on recombinant GABAA receptors. The aim of this study was to examine the effects of sevoflurane and desflurane on potentiation of GABA-induced responses in the wild-type GABAA receptor and in receptors mutated in TM2 of the alpha1, alpha 2, or beta 2 subunits. Methods GABAA receptor alpha 1 or alpha 2, beta 2 or beta 3, and gamma 2s subunit cDNAs were expressed for pharmacologic study by transfection of human embryonic kidney 293 cells and assayed using the whole cell voltage clamp technique. Concentration-response curves and EC50 values for agonist were determined in the wild-type alpha 1 beta 2 gamma 2s and alpha 2 beta 3 gamma 2s receptors, and in receptors harboring mutations in TM2, such as alpha1(S270W)beta 2 gamma 2s, alpha 1 beta 2(N265W)gamma 2s, and alpha2(S270I)beta 3 gamma 2s. The actions of clinically relevant concentration of volatile anesthetics (isoflurane, sevoflurane, and desflurane) on GABA activated Cl- currents were compared in the wild-type and mutant GABAA receptors. Results Both sevoflurane and desflurane potentiated submaximal GABA currents in the wild-type GABAA alpha 1 beta 2 gamma 2s receptor and alpha 2 beta 3 gamma 2s receptor. Substitution of Ser270 in TM2 of the alpha subunit by a larger amino acid, tryptophan (W) or isoleucine (I), as in alpha1(S270W)beta 2 gamma 2s and alpha 2(S270I)beta 3 gamma 2s, completely abolished the potentiation of GABA-induced currents by these anesthetic agents. In contrast, mutation of Asn265 in TM2 of the beta subunit to tryptophan (W) did not prevent potentiation of GABA-induced responses. The actions of sevoflurane and desflurane in the wild-type receptor and in mutated receptors were qualitatively and quantitatively similar to those observed for isoflurane. Conclusions Positions Ser270 of the GABAA alpha1 and alpha2 subunits, but not Asn265 in the TM2 of the beta2 subunit, are critical for regulation of the GABAA receptor by sevoflurane and desflurane, as well as isoflurane, consistent with the idea that these three volatile anesthetics share a common site of actions on the alpha subunit of the GABAA receptor.
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Zhang, J. T. "Sequence requirements for membrane assembly of polytopic membrane proteins: molecular dissection of the membrane insertion process and topogenesis of the human MDR3 P-glycoprotein." Molecular Biology of the Cell 7, no. 11 (1996): 1709–21. http://dx.doi.org/10.1091/mbc.7.11.1709.
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The biogenesis of membrane proteins with a single transmembrane (TM) segment is well understood. However, understanding the biogenesis and membrane assembly of membrane proteins with multiple TM segments is still incomplete because of the complexity and diversity of polytopic membrane proteins. In an attempt to investigate further the biogenesis of polytopic membrane proteins, I used the human MDR3 P-glycoprotein (Pgp) as a model polytopic membrane protein and expressed it in a coupled cell-free translation/translocation system. I showed that the topogenesis of the C-terminal half MDR3 Pgp molecule is different from that of the N-terminal half. This observation is similar to that of the human MDR1 Pgp. The membrane insertion properties of the TM1 and TM2 in the N-terminal half molecule are different. The proper membrane anchorage of both TM1 and TM2 of the MDR3 Pgp is affected by their C-terminal amino acid sequences, whereas only the membrane insertion of the TM1 is dependent on the N-terminal amino acid sequences. The efficient membrane insertion of TM3 and TM5 of MDR3 Pgp, on the other hand, requires the presence of the putative TM4 and TM6, respectively. The TM8 in the C-terminal half does not contain an efficient stop-transfer activity. These observations suggest that the membrane insertion of putative TM segments in the human MDR3 Pgp does not simply follow the prevailing sequential event of the membrane insertion by signal-anchor and stop-transfer sequences. These results, together with my previous findings, suggest that different isoforms of Pgp can be used in comparison as a model system to understand the molecular mechanism of topogenesis of polytopic membrane proteins.